Because CASQ2 and RyR2 are involved in different aspects of the excitation-contraction coupling process, we hypothesized that these mutations are associated with different functional and intracellular Ca²+ abnormalities. To test the hypothesis we generated induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CM) from CPVT1 and CPVT2 patients carrying the RyR2R420Q and CASQ2D307H mutations, respectively, and investigated in CPVT1 and CPVT2 iPSC-CM (compared to control): (i) The ultrastructural features; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca2+]i transient characteristics. Our major findings were: (i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased diastolic [Ca2+]i. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2 syndromes.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia characterized by syncope and sudden death occurring during exercise or acute emotion. CPVT is caused by abnormal intracellular Ca CASQ2 is a high-capacity, low affinity Ca²+ binding protein, operating as a major Ca²+ buffering factor. Since both proteins are major players in (E-C) coupling, the functional derangements in intracellular Ca²+ handling resulting from the mutated RyR2 and CASQ2 may cause delayed afterdepolarizations (DADs) and triggered arrhythmias which constitute the electrophysiological mechanism underlying CPVT e.g. D307H mutation, and to demonstrate that the mutated cardiomyocytes generated DADs and triggered arrhythmia in response to β-adrenergic stimulation + handling abnormalities. We tested the hypothesis by comparing in CPVT1 and CPVT2 iPSC-CM: (i) The ultrastructure; (ii) the effects of isoproterenol, caffeine and ryanodine on the [Ca2+]i transient characteristics. Briefly, the mutated cardiomyocytes were less developed untrastructurally than control, and the responses to isoproterenol, caffeine and ryanodine were different than control. These findings demonstrate that the mutated iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2.Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder characterized by episodic syncope and sudden death occurring during exercise. CPVT is caused by abnormal Cagene CPVT or the agene CPVT or the aR420Q mutation and from 4 CPVT2 patients carrying the CASQ2D307H mutation, as described in the Supplement. Of the 4 CPVT patients, 2 patients  were included in our previous study Skin biopsies were obtained from a 25-years old CPVT1 woman (HDF15) carrying RyR22+]i transients were measured from small contracting areas of EBs by means of fura-2 fluorescence, and analysed as described in the i we utilized three protocols to decipher the differences/similarities in the intracellular Ca2+ abnormalities in the mutated cardiomyocytes: β-adrenergic stimulation, exposure to caffeine and to ryanodine.Because CPVT is an inherited arrhythmia resulting from increased cytoplasmic [Ca2+]i transients in control versus mutated iPSC-CM served as an important experimental tool to decipher intracellular Ca2+ abnormalities in the mutated cardiomyocytes. As shown by the representative experiments  isoproterenol increased (as expected) the [Ca2+]i transient amplitude, the maximal rate of rise and decay , while diastolic [Ca2+]i was unchanged. In contrast, in CPVT1 and CPVT2 iPSC-CM isoproterenol caused three different types of effects: (i) ‘No-response’ to isoproterenol; (ii) ‘Arrhythmias’ and (iii) ‘Intracellular Ca2+ rise’ associated with diminished [Ca2+]i transient amplitude. Hence, while the effects of isoproterenol in healthy iPSC-CM are those reported by us and others in previous studies i) No-response to isoproterenol: 59% in CPVT1 iPSC-CM (composed of 43% from clone 15.4 and 57% from clone 15.1); 37% in CPVT2 iPSC-CM  Arrhythmias: 16% in CPVT1 iPSC-CM clone 15.1; 37% in CPVT2 iPSC-CM (composed of 58% from clone 12.4), 14% from clone 12.12, 14% from clone 19S1 and 14% in clone 7.5. (iii) Marked intracellular Ca2+ rise associated with diminished [Ca2+]i transient amplitude: 25% in CPVT1 iPSC-CM clone 15., and 26% in CPVT2 iPSC-CM . Collectively, in both CPVT1 and CPVT2 iPSC-CM, in the ‘No-response’ group none of the [Ca2+]i transient characteristics was affected, and arrhythmias were not generated. In the ‘Arrhythmias’ group none of the [Ca2+]i transient characteristics was affected  the [Ca2+]i transient amplitude, +d[Ca2+]i/dt and −d[Ca2+]i/dt.Investigating the effects of isoproterenol on the i transient amplitude. Within 20 sec. after intracellular Ca2+ peaked, it declined and the [Ca2+]i transients attained a steady state level close to the their pre-caffeine amplitude. Similar results are shown for clone 24.5 in Figure S2A. In CPVT1 cardiomyocytes clone 15.1  The per cent change in caffeine-induced Ca2+ signal amplitude, compared to the pre-caffeine [Ca2+]i transient amplitude. (ii) The per cent change in the area of the caffeine-induced Ca2+ signal, compared to the pre-caffeine [Ca2+]i transient area. Hence, compared to control cardiomyocytes, both measures of the caffeine response were smaller (P < 0.05) in CPVT1 cardiomyocytes and larger (P < 0.05) CPVT2 cardiomyocytes  ryanodine decreased the [Ca2+]i transient amplitude (−26.3%), +d[Ca2+]i/dt (−29.4%) and −d[Ca2+]i/dt (−15.3%) (intracellular [Ca2+]i was unaffected), the effect on CPVT1 (n = 5) and CPVT2 cardiomyocytes (n = 8) was much more pronounced. In both CPVT1 and CPVT2 cardiomyocytes, ryanodine caused marked elevation in [Ca2+]i, along with reduction of [Ca2+]i transient amplitude , +d[Ca2+]i/dt (−51.9 and −52.8 respectively) and −d[Ca2+]i/dt .Next, we determined the effects of 10 μM ryanodine on the Caa2+i tran2+]i-overload, and since CASQ2 and RyR2 participate in different aspects of the E-C coupling, we hypothesized that mutations in these genes might be associated with distinct disturbances in the intracellular Ca2+ handing machinery. Whereas several studies i) Ultrastructurally, CASQ2 and RyR2 mutated cardiomyocytes were less developed than control cardiomyocytes, and had a slower spontaneous firing rate. (ii) While in control iPSC-CM isoproterenol caused positive inotropic and lusitropic effects, in the mutated cardiomyocytes isoproterenol was either ineffective, caused arrhythmias, or markedly increased intracellular Ca2+, along with negative inotropic and lusitropic effects. Importantly, positive inotropic and lusitropic effects were not induced in mutated cardiomyocytes. (iii) The effects of caffeine and ryanodine in mutated cardiomyocytes differed from control cardiomyocytes. Our results show that iPSC-CM are useful for investigating the similarities/differences in the pathophysiological consequences of RyR2 versus CASQ2 mutations underlying CPVT1 and CPVT2, respectively.Catecholaminergic polymorphic ventricular tachycardia is a malignant inherited arrhythmia caused by mutations in the RyR2 and CASQ2 genes. Because CPVT is related to diastolic i transient characteristics were unaffected. In the third group (25% in CPVT1 and 26% in CPVT2) isoproterenol markedly increased diastolic [Ca2+]i, along with a pronounced decline in +d[Ca2+]i/dt and −d[Ca2+]i/dt, corresponding to negative inotropic and lusitropic effects, respectively. While a single mechanism is unlikely to account for the three different responses to isoproterenol in the mutated cardiomyocytes  Augmented diastolic SR Ca2+ leak diminishes SR Ca2+ reservoir, and therefore less RyR2-mediated Ca2+ efflux into the cytoplasm. (ii) Ca2+-induced inhibition of the L type Ca2+ current  Ca,L density, thereby attenuating [Ca2+]i transient amplitude and +d[[Ca2+]i/dtmax. (iii) The excessive rise in intracellular Ca2+ diminishes the Ca2+ gradient across the SR membrane and therefore the driving force for Ca2+ efflux into the cytoplasm, which in turn will attenuate the [Ca2+]i transient. The emanating question from these findings is why isoproterenol generated three distinct phenotypes in a seemingly similar population of EBs. We hypothesize that these diverse effects could be related to the heterogeneous composition of the EBs regarding the ratio between nodal, atrial and ventricular cardiomyocytes, including Purkinje fibres. In this regards, based on optical mapping in isolated Langendorff-perfused RyRR4496C/WT CPVT mouse hearts, Jalife’s group suggested that CPVT arrhythmias originate from the Purkinje fibres 2+ handling characteristics, which promote spontaneous Ca2+ release and triggered arrhythmias 2+ was unaffected, and in cells where Ca2+ levels were elevated, arrhythmias were not generated. This distinction between the two effects of isoproterenol suggests that the triggered beats originated delayed afterdepolarizations (DADs) caused by spontaneous diastolic SR Ca2+ release  and not due to cytoplasmic Ca2+ overload although this phenomena does occur in CPVT cardiomyocytes.In the third group in which isoproterenol increased intracellular Caated Fig.. Three p2+ stores in CPVT cardiomyocytes, and distinguished between CPVT1 and CPVT2. In brief, while in CPVT1 cardiomyocytes caffeine released less Ca2+ (represented by the change in [Ca2+]i transient amplitude and area) than in control cardiomyocytes, in CPVT2 cardiomyocytes the release of Ca2+ from intracellular stores was markedly augmented  et al. reported that caffeine-induced Ca2+ transients generated smaller NCX current in CPVT1 iPSC-CM (RyR2F24831), compared to control, reflecting their smaller Ca2+ stores 2+ storage capacity as well as the mutation phenotype. Accordingly, we suggest that the massive and prolonged Ca2+ release in CPVT2 cardiomyocytes originated from high levels of free SR Ca2+ due to the mutated CASQ2 protein [e.g. 3], and not necessarily from a total higher SR Ca2+ levels. In contrast, the attenuated caffeine-induced Ca2+ release in CPVT1 cardiomyocytes is probably due to the mutated RyR2, which is likely to have low sensitivity to caffeine along with reduced SR Ca2+ storage, as suggested by the ultrastructural analysis in which CPVT1 iPSC-CM presented decreased SR peripheral width (−15%) compared to control cardiomyocytes.The caffeine and ryanodine experiments further elucidated the abnormal functionality of internal Canted Fig.. Further RyR2P232S et al. 2+]i transient amplitude and area , in agreement with previous studies in iPSC-CM and hESC-CM 2+]i. In contrast, in CPVT cardiomyocytes ryanodine markedly elevated diastolic [Ca2+]i along with pronounced decline in the [Ca2+]i transient characteristics. These results can be explained based on the scheme shown in Figure2+ release, in the mutated cardiomyocytes, due to altered sensitivity of the deregulated RyR2, ryanodine at 10 μM acts as an agonist and releases SR Ca2+ into the cytoplasm.In control cardiomyocytes 10 μM ryanodine attenuated the [CaR420Q and CPVT2-CASQ2D307H patients, which gave rise to functional mutated cardiomyocytes exhibiting the disease-specific arrhythmogenic characteristics. This experimental system enables us to recapitulate in vitro the features of human genetic disorders, such as CPVT, in which the disease mechanisms can be explored and candidate drugs screened and developed in the physiologic and disease-causing settings on a patient-specific level. Indeed, both CPVT1 and CPVT2 cardiomyocytes generated arrhythmias in response to β-adrenergic stimulation and displayed Ca²+ abnormalities in response to ryanodine. The exposure of the mutant iPSC-CM to caffeine revealed the different intracellular Ca²+ abnormalities between CPVT1 and CPVT2 cardiomyocytes. Collectively, our results support the notion that the propensity to develop arrhythmias in CPVT is associated with disturbed intracellular Ca2+ handling, SR load and storage capacity.In summary, for the first time we compared iPSC from CPVT1-RyR2The author confirm that there are no conflicts of interest.


We report here that some, but not all, TAS2R agonists decrease [Ca2+]i and relax HASM contracted by G-protein coupled receptors (GPCRs) that stimulate [Ca2+]i. This suggests both a second pathway by which TAS2Rs relax, and, a heterogeneity of the response phenotype. We utilized eight TAS2R agonists and five procontractile GPCR agonists in cultured HASM cells. We find that heterogeneity in the inhibitory response hinges on which procontractile GPCR is activated. For example, chloroquine inhibits [Ca2+]i increases from histamine, but failed to inhibit [Ca2+]i increases from endothelin-1. Conversely, aristolochic acid inhibited [Ca2+]i increases from endothelin-1 but not histamine. Other dichotomous responses were found when [Ca2+]i was stimulated by bradykinin, angiotensin, and acetylcholine. There was no association between [Ca2+]i inhibition and TAS2R subtype, nor whether [Ca2+]i was increased by Gq- or Gi-coupled GPCRs. Selected studies revealed a correlation between [Ca2+]i inhibition and HASM cell-membrane hyperpolarization. To demonstrate physiologic correlates, ferromagnetic beads were attached to HASM cells and cell stiffness measured by magnetic twisting cytometry. Consistent with the [Ca2+]i inhibition results, chloroquine abolished the cell stiffening response (contraction) evoked by histamine but not by endothelin-1, while aristolochic acid inhibited cell stiffening from endothelin-1, but not from histamine. In studies using intact human bronchi, these same differential responses were found. Those TAS2R agonists that decreased [Ca2+]i, promoted hyperpolarization, and decreased HASM stiffness, caused relaxation of human airways. Thus TAS2Rs relax HASM in two ways: a low-efficiency de novo [Ca2+]i stimulation, and, a high-efficiency inhibition of GPCR-stimulated [Ca2+]i. Furthermore, there is an interaction between TAS2Rs and some GPCRs that facilitates this [Ca2+]i inhibition limb.Asthma is characterized by airway inflammation and airflow obstruction from human airway smooth muscle (HASM) constriction due to increased local bronchoconstrictive substances. We have recently found bitter taste receptors (TAS2Rs) on HASM, which increase [Ca We proposed that upon activation with contractile GPCR agonists, the TAS2R signaling from this pathway will antagonize cell membrane depolarization, promote a decrease in single cell stiffness, and cause relaxation of human airways. Based on the heterogeneity of the [Ca2+]i-inhibition response observed in initial studies, we further hypothesized that the capacity for TAS2Rs to inhibit [Ca2+]i and relax ASM (by this mechanism), is dependent on which GPCR is acting to stimulate [Ca2+]i and contract the airway. Thus in the current work we utilized multiple TAS2R agonists for the three highest expressing TAS2R subtypes in HASM, and agonists for multiple Gq-and/or Gi-coupled GPCRs which increase [Ca2+]i. We indeed demonstrate a dual mechanism for TAS2R signaling, where the inhibitory pathway hinges upon how [Ca2+]i is increased in the ASM cell.These issues have led to our search for other drug targets that promote human ASM (HASM) relaxation . We founlization . Intracelization . This simembrane . Such demembrane , and thumembrane ,20. Of nmembrane . Howevermembrane ,12,21–24membrane –16 have neration ,26. We hract ASM . In studagonists . We hypohttp://ndriresource.org) as described i recorded over 120 sec (unless indicated otherwise) using the FlexStation3 plate reader (Molecular Devices). Fluorescence  was measured every 1.52 sec. Baseline fluorescence background (Fo) was captured for 16–19 sec before the addition of procontractile agonist plus or minus TAS2R agonists . When present, the final concentration of DMSO or DMF was below 0.25%, a concentration that did not affect the baseline signal. Calcium response  was calculated by subtracting basal fluorescence signal  from the agonist peak value of fluorescence signal (F). The effect of co-stimulation with TAS2R agonists on [Ca2+]i response evoked by procontractile agonists was expressed as percentage of ΔF in the presence (ΔFa) of the TAS2R agonist relative to the ΔF in the absence (ΔF0) of the TAS2R agonist [100 x (1- ΔFa / ΔF0)].For measurement of i in HASM cells. We first built upon our previous findings by determining the EC50 for each TAS2R agonist for stimulating [Ca2+]i in HASM cells . Co-treatment of cells with 3 μM histamine and increasing concentrations of the TAS2R10 agonist CQ showed a dose-dependent decrease in the peak [Ca2+]i beginning with 1 μM CQ and up to concentrations of ~1 mM CQ (2+]i was observed (2+]i response to CQ was evident in the presence of histamine stimulation (50 = 14.8 ± 5.8 μM (N = 4)  . Of note ~450 μM . Togethe2+]i elicited by histamine. 2+]i evoked by histamine. The magnitude of the [Ca2+]i peaks remained unchanged up to ~200 μM AA, above which an increase in the signal was detected. Similar to AA, co-treatment of HASM cells with SAC, a bitter tastant that activates TAS2R31, did not inhibit [Ca2+]i stimulated by histamine . As expected, AA in the absence of ET-1 stimulates [Ca2+]i . Quantitative analysis revealed that CQ, STRY, YOH and QUI inhibited [Ca2+]i evoked by histamine while AA, FFA, and SAC had no effect . We considered that the heterogeneity in the inhibitory response could be due to whether [Ca2+]i was stimulated by a Gq—vs a Gi-coupled receptor. Of note, pertussis toxin treatment cannot be utilized for this differentiation, since it also inactivates the TAS2R G-protein gustducin i mobilization phenyl)histamine , was also inhibited by CQ. Importantly, the Gi-coupled HRH3 agonist immethridine-stimulated [Ca2+]i was inhibited by CQ to a similar extent. To further assess the possibility of selectivity for Gq- vs Gi-coupled receptors for TAS2R-mediated inhibition, we exposed cells to 400 μM somatostatin, which activates the four somatostatin subtypes, all of which couple to Gi but not to Gq. As shown in 2+]i stimulation by somatostatin was also inhibited by TAS2R agonists to ~50%. Taken together, these data indicate that regardless of whether the GPCR couples to Gq or Gi, TAS2R activation can reduce elevated [Ca2+]i, and thus the heterogeneity that we observe between contractile GPCR agonists and TAS2R agonists cannot be readily attributed to this mechanism.ustducin . We thuslization . N-Methy2+]i induced by procontractile agonists promotes cell membrane depolarization and actin-myosin activation, ultimately leading to contraction. Conversely, hyperpolarization of the cell membrane antagonizes contraction. One of the proposed mechanisms by which TAS2R activation induces smooth muscle relaxation is a [Ca2+]i-dependent decrease in plasma membrane potential (hyperpolarization). We therefore explored the changes in membrane potential that occurred upon stimulation of HASM cells with bronchoconstrictive agonists in the presence and absence of co-administration of TAS2R agonists. We used a validated assay in which an increase or decrease in probe fluorescence is indicative, respectively, of depolarization or hyperpolarization of the cell membrane. Exposure of HASM to 60 mM KCl resulted in the expected depolarization . As shown in 2+]i by ~70%, fully blocked the histamine-mediated increase in cell stiffness  and by measuring force in intact human bronchi (which ascertains the effect on coordinated smooth muscle function of the intact airway). The heterogeneity observed for certain pairs for inhibiting [Ca2+]i was indeed recapitulated in measurements of stiffness in single cells. Cell stiffness, which was increased by the procontractile agonists was antagonized only by TAS2R agonists that decreased [Ca2+] (and opposed membrane depolarization) under the same conditions. Finally, intact human airway responses were consistent with the results from the [Ca2+]i, membrane potential, and isolated cell mechanics studies. Taken together, these findings demonstrate the relevance of this TAS2R pathway to human physiologic responses.As introduced earlier, TAS2Rs expressed on HASM cells represent a novel target for a new class of direct bronchodilators for the treatment of obstructive lung diseases such as asthma and chronic obstructive lung disease. Here we report the function of eight TAS2R agonists acting on HASM under physiologically relevant conditions of exposure to procontractile GPCR agonists. We hypothesized that TAS2R activation decrease [Ca2+]i as a key intracellular event leading to ASM relaxation [2+]i in isolated HASM cells, that was blocked by Gβγ and PLC inhibitors, and substantially attenuated by an IP3 receptor antagonist. In addition, human and mouse ASM have been shown to express gustducin  from these stores into an intracellular space. However, we found that the temporal and spatial distribution of this Ca2+ in HASM cells was suggestive of the activation of one or more cell surface channels such as the large conductance calcium dependent K+ channel (BKCa), which leads to ASM cell hyperpolarization. There appears to be additional mechanisms by which this specialized pool of [Ca2+]i evoked from TAS2R activation leads to relaxation [2+]i also fail to relax [2+]i with EC50 values in the high μM to mM range in HASM  in the presence of AA, which causes no change in [Ca2+]i or relaxation  and to Gαs (stimulating cAMP) i stimulation and inhibition are not readily apparent. This phenomenon is made even more complex by the heterogeneity of the inhibitory response, which is dependent upon which GPCR is providing the [Ca2+]i stimulation.There is considerable precedence for receptors within the GPCR superfamily (of which the TAS2R family is a member) coupling to multiple pathways, including those with competing or opposing effects. For example, the αng cAMP) . The latng cAMP) ,41. Specs or Gαi ,43, thustivation . GPCR sitivation ,45,46. Ttivation –49. The 2+] with high agonist concentrations which results in membrane hyperpolarization and HASM relaxation. In addition, when TAS2Rs are activated by lower concentrations of agonist under conditions of elevated [Ca2+] by various bronchoconstrictors, they inhibit this [Ca2+]i increase, oppose membrane depolarization, and thus relax precontracted HASM. The inhibitory response is dependent on which GPCR is acting to stimulate [Ca2+] mobilization. This suggests a compartmentalization of [Ca2+]i signals in HASM, of which some are accessible to TAS2Rs, or, other forms of signaling interactions between TAS2Rs and bronchoconstrictive receptors.In conclusion, TAS2Rs on HASM are recognized to stimulate i mobilization measured in response to 3 μM histamine, 1 μM ET-1, or 1 μM ionomycin. The responses to histamine and ET-1 (as well as ionomycin) were no different in cells pretreated with CQ or AA, compared to pretreatment with buffer alone, indicated a reversal of TAS2R agonist effect. Data is from 4–5 experiments performed in triplicate.40,000 HASM cells/well were exposed to buffer alone (representing “untreated”) or buffer with 50 μM CQ or AA for 5 min. Cells were washed twice with PBS, and then [Ca(TIFF)Click here for additional data file.


Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca2+ release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca2+ release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca2+ release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca2+ release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca2+ release by 86% . In RyR3-KO, NAADP-evoked Ca2+ release reduced by ∼50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca2+ release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca2+ liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca2+ release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca2+ release from the all stores is RyR1 > TPC2 > RyR3 > TPC1 >> RyR2. However, when assessing NAADP-induced Ca2+ release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca2+ liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca2+ release via TPCs from endosomes/lysosomes triggers the detectable Ca2+-induced Ca2+ release via RyR1 and RyR3 occurring from the granules and the ER.Intracellular Ca Early work on salivary glands provided evidence for Ca2+ pump-mediated Ca2+ uptake into intracellular stores 2+ from such stores 2+ signalling studies on pancreatic acinar cells led to many important findings including the discovery of inositol trisphosphate (IP3) as a messenger releasing Ca2+ from intracellular stores 2+ signal generation in the apical granular pole of the cells 2+ tunnels 3 induces responses from the endoplasmic reticulum ER 3 receptors (IP3Rs) are located in the ER including the apical area of acinar cells 3 can release Ca2+ from a different organelle containing a vacuolar H+-ATPase 3-evoked Ca2+ release from bovine adrenal medullary secretory vesicles 3 and cADPR-induced Ca2+ release from this organelle 2+2+ spikes regulating exocytotic secretion of digestive enzymes are mainly due to Ca2+ release from the ER strands in the secretory granular area 2+ inside the ER throughout the cell 2+ from the acid stores may be of particular significance for the initiation of acute pancreatitis 2+ releasing messengers IP3, cADPR and NAADP 3Exocrine gland cells have provided the most important models for elucidating the mechanisms underlying hormone- or neurotransmitter-evoked intracellular Ca2+ signalling 2+ release from the lysosomes. Calcium-induced calcium release (CICR) via IP3 or ryanodine receptors has been proposed as an explanation for the subsequent amplification of the response by Ca2+ release from the ER 2+ release in both the secretory granule stores and the ER 2+ release from endosome/lysosome stores in pancreatic acinar cells would be extremely difficult to detect due to the small size of the endo/lysosomal Ca2+ store 2+ signalling are very unclear 2+ release in pancreatic acinar cells.There are conflicting hypotheses about the mechanism underlying NAADP-induced Ca22.1Most of the reagents were purchased from Sigma (UK). Ned-19 was obtained from Tocris (UK). Thapsigargin and ATP were from Merck Millipore (UK). Rabbit anti-TPC1 and anti-TPC2 polyclonal antibodies were purchased from Lifespan BioSciences (UK). Rabbit anti-RyRs polyclonal antibodies were obtained from Merck Millipore (UK).2.22, 1; glucose 10; CaCl2 1; pH 7.2. Cells were then loaded with an appropriate fluorescent dye (Fluo-5N AM or Fluo-4 AM) following the manufacturer's instruction. All experiments were carried out at room temperature using freshly isolated cells, attached to the poly-L-lysine coverslips of the perfusion chamber.Pancreatic acinar cells  were isolated from the pancreas of adult C57BL/6 mice  as described previously 2.32, 1; EGTA, 0.1; CaCl2 0.05; pH 7.2.Two-photon permeabilization was performed as described previously 2.4t-test was used .To enable comparison, confocal images were recorded using identical laser power, photomultiplier sensitivity, and were processed using identical values for contrast and brightness. Statistical significance for the comparison between two groups of data Student's 33.12+ responses elicited by physiological concentrations of CCK can be specifically blocked by inactivation of the NAADP receptor, while responses to ACh were unaffected 2+ signal generation. In a previous study, Ned-19, at a high concentration (100 μM), was shown to block NAADP-mediated Ca2+ release and binding to the NAADP receptor 2+ responses induced by 5 pM CCK  inhibited NAADP-elicited Caby 67.8% C–E and I67.8%  inhibition of the NAADP-elicited Ca2+ release by 85.7%  , the NAADP-elicited release of Cawt cells G. The NA control G. Howeve by 94%) G.2+-release in rat astrocytes and in the sea urchin egg 3 receptors 2+ signals by 75.8% . IP3 (10 μM)- or cADPR (10 μM)-induced Ca2+ responses were not reduced in the presence of 20 μM PPADS.It has been reported that the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) can significantly inhibit NAADP-induced Ca3.32+ signalling, we have employed permeabilized pancreatic acinar cells isolated from RyR3 KO mice. The NAADP (100 nM) elicited Ca2+ release from the internal stores of RyR3 KO cells was reduced to about 48% of the control value obtained from wt cells  (2+ release by only 49% (3 (10 μM) or thapsigargin (10 μM) in cells from RyR3 KO animals were not significantly different from responses in wt cells (To test for a role of RyRs in NAADP-induced Caibition) C and E. only 49% D and E. wt cells E.n = 5) as compared to control . Subsequent application of 10 μM IP3 resulted in a substantial Ca2+ release from the internal stores showing that the stores were duly loaded and capable of Ca2+ release . The Ca2+ release elicited by cADPR (10 μM) in permeabilized cells pre-incubated with the RyR1 antibody was only modestly decreased  as compared to control  (SP1G).To test the involvement of different RyR sub-types in NAADP signalling, we used pre-incubation of permeabilized cells with antibodies against RyR1, 2 and 3. Supplemental Figures SP1A and SP1G show that pre-treatment of cells with antibodies against RyR1 dramatically decreased the responses to NAADP (100 nM) , but the cADPR responses were substantially reduced  . The RyR3 antibody partially reduced both cADPR and NAADP responses .Incubation of permeabilized cells with the RyR2 antibody did not significantly affect the magnitude of the NAADP-elicited Can = 5 for NAADP; 3.6 ± 0.6%, n = 11 for cADPR) (SP1G). The largest inhibitory effect on NAADP-elicited Ca2+ release  was achieved when permeabilized cells were pre-treated with a combination of antibodies against RyR1 and RyR3  (SP1G). In the same set of experiments cADPR-induced responses were only partially inhibited  (SP1G) whereas IP3-elicited Ca2+ release was unaffected by the RyRs antibodies (SP1G).Combination of antibodies against RyR1 and RyR2 markedly inhibited the NAADP and cADPR responses  . These data suggest that both NAADP- and cADPR-elicited Ca2+ signalling are dependent on functioning RyR channels in pancreatic acinar cells. As shown in Figure SP1G, the inhibition of RyR types 1 and/or 3 exhibits the most effective decrease of NAADP-induced signalling while inhibition of RyR2 has no effect. All three types of RyRs are involved in cADPR responses with RyR2s being the most important. IP3 (10 μM) in the presence of RyR and/or TPEN (200 μM) were used as a control of Ca2+ release from internal Ca2+ stores (SP1G).A mixture of antibodies against all three types of RyRs almost completely prevented both NAADP – and cADPR-induced Ca2+ release. By analysing the distribution of RyR type-dependent responses to 100 nM NAADP or cADPR in cells from wt and RyR3 KO mice in the presence or in the absence of RyR type specific antibodies  2+ release from IP3Rs and RyRs, this is possible in permeabilized cells 2+ release elicited by NAADP, but not for Ca2+ liberation evoked by cADPR or IP3  being amplified very substantially by further Ca2+ release from both larger acid stores (zymogen granules) and the ER. This interpretation fits well with the previous finding that NAADP-elicited Ca2+ release from acid stores is blocked by clamping the cytosolic Ca2+ concentration at the normal resting level, whereas this is not the case for Ca2+ release evoked by cADPR and IP3 (Fig. 6 in 2+ release from acid stores is completely dependent on functional TPCs (2+ spiking Our new findings make it necessary to revise the current model nal TPCs G. In thi2+ release is completely dependent on RyRs (Some of our new findings do, however, require some additions to the simple model outlined above. It is clear that whereas both NAADP- and cADPR-elicited Ca on RyRs , the sub on RyRs , and req3Rs and RyRs are both present in acid pools and the ER and that all 3 intracellular Ca2+ releasing messengers can liberate Ca2+ from both pools 2+ release from all the intracellular stores is not totally dependent on TPCs (2+ release from acid stores can be blocked by clamping the cytosolic Ca2+ concentration at 100 nM, this is not the case for NAADP-elicited Ca2+ release from all the stores 2+ release that is independent of TPCs because, in the intact cells, Ned-19 completely blocked cytosolic Ca2+ spiking evoked by a physiological concentration of CCK (2+ release from all the stores was markedly reduced but not abolished by Ned-19 (2+ release from all stores in the absence of functional TPCs (2+ release from the acid store (We have previously reached the conclusion that IP on TPCs F, as is  on TPCs G, may inn of CCK . In the y Ned-19  in agreenal TPCs F and in id store G.2+ signalling in pancreatic acinar cells, but also reveal a more complex pattern of potential interactions between different Ca2+ release channels in various compartments that require further investigation.Overall, our data provide critical evidence for a central role of TPCs, and particularly TPC2, in NAADP-elicited CaJVG, RMC and PEF did the experimental work and analyzed the data together with OVG. KM and MWS organized experimental work with RyR3-KO. JP provided TPC2–KO. OHP wrote the paper together with OVG and JVG.The authors declare that they have no conflict of interest.


Endogenous and exogenous signals induce cytoplasmic Ca2+ ([Ca2+]cyt) elevation, which are responsible for the appropriate downstream responses.Ca2+]cyt elevation in response to exudate preparations from the pathogenic mibrobes Alternaria brassicae, Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The cytoplasmic Ca2+elevation mutant1 (cycam1) is susceptible to infections by A. brassicae, its toxin preparation and sensitive to abiotic stress such as drought and salt. It accumulates high levels of reactive oxygen species and contains elevated salicylic acid, abscisic acid and bioactive jasmonic acid iso-leucine levels. Reactive oxygen species- and phytohormone-related genes are higher in A. brassicae-treated wild-type and mutant seedlings. Depending on the analysed response, the elevated levels of defense-related compounds are either caused by the cycam mutation and are promoted by the pathogen, or they are mainly due to the pathogen infection or application of pathogen-associated molecular patterns. Furthermore, cycam1 shows altered responses to abscisic acid treatments: the hormone inhibits germination and growth of the mutant.Here we report on an ethyl-methane sulfonate-mediated Arabidopsis mutant that fails to induce [Ca2+]cyt elevation in response to exudate preparations from various microbes. The higher susceptibility of the mutant to pathogen infections correlates with the higher accumulation of defense-related compounds, such as phytohormones, reactive oxygen-species, defense-related mRNA levels and secondary metabolites. Therefore, CYCAM1 couples [Ca2+]cyt elevation to biotic, abiotic and oxidative stress responses.We isolated an Arabidopsis mutant which fails to induce [Ca Ca2+. Ca2+2+ n plants ,8. Underus level ,10. In pchannels -11. H+/C2+]cyt elevation is one of the earliest physiological events in root and leaf cells in response to pathogenic stimuli. Upon perception of signals from pathogenic fungi or/and their pathogen-associated molecular patterns (PAMPs), [Ca2+]cyt levels transiently increase in the host cells within seconds [2+]cyt elevation which leads to the activation of defence-signalling cascades against the attempted pathogen invasion cyt elevation in response to exudate components from Alternaria brassicae (Berk.) Sacc. A. brassicae is a necrotrophic deuteromycete fungus which causes black spot disease in crucifers including A. thaliana. It is a seed-, air- and soil-borne fungus that penetrates through all plant parts and causes lesions on leaves, stems, siliques and roots cyt elevation in Arabidopsis stably expressing the Ca2+ reporter protein aequorin. We have isolated and characterized a cytosolic calcium elevation mutant1 (cycam1) which does not induce [Ca2+]cyt elevation in response to the non-toxic exudate components. Further characterization of cycam1 demonstrated that it also fails to induce [Ca2+]cyt elevation in response to exudate preparations from Rhizoctonia solani, Phytophthora parasitica var. nicotianae and Agrobacterium tumefaciens. The mutant is susceptible to infection by A. brassicae and sensitive to abscisic acid (ABA), drought and salt stress. Thus, the mutated gene couples [Ca2+]cyt elevation to biotic and abiotic stress responses.We demonstrate that besides these Toxs, non-toxic low molecular weight exudates components from A. thaliana roots in the Col-0 background (pMAQ2) [2+]cyt values of 70 ± 0,6 nM (n = 16). A rapid and transient increase in the [Ca2+]cyt concentration is observed 40 sec after the application of a cell wall extract (CWE), a water-diffusible exudate preparation from mycelia (EPM), germinating spores (EPS) or a Tox preparation from A. brassicae to the roots  ,28 gave 6 nM n = . A rapidA. brassicae exudates and Tox preparations showed very similar [Ca2+]cyt elevation kinetics which did not change after heat treatment (20 min at 121°C by autoclaving) indicating that the components are thermostable  mutagenesis of transgenic apoaequorin-carrying M1 seeds in the Col-0 background [2+]cyt level for 1 min, the response to the CWE was measured for 10 min. Roots which did not respond to the stimulus were used for the total discharge reaction to ensure that the lack of [Ca2+]cyt elevation is not caused by a mutation in the apoaequorin gene. Screening of approximately 150.000 individual M2 plants identified 12 mutants which completely failed to induce [Ca2+]cyt elevation in response to the CWE; they were named cytoplasmic calcium elevation mutants (cycam)  or WT (La), [Ca2+]cyt elevation to the CWE was restored in ~25% of F2 progenies, indicating that the mutations are recessive.96-well plates in combination with a plate-reader luminometer equipped with an automatic injection system were used to screen for Arabidopsis mutants which do not show cyt response was induced by the four A. brassicae-derived preparations. 5 μM staurosporine was used, because the basal level of [Ca2+]cyt and the total aequorin discharge was not changed at this concentration [cf. 32]. Application 1 h prior to the treatment with one of the four Ca2+-inducing stimuli significantly reduced [Ca2+]cyt elevation . Therefore, CYCAM1 is involved in [Ca2+]cyt elevations in response to different, but not all microbes.Since a hyalina induced 2+]cyt responses induced by the CWEs and EPMs from these four microbes show a refractory behaviour, roots of WT and the two cycam1 alleles were challenged first with the CWE from A. brassicae and subsequently with either the CWE or EPM from one of the other microbes. The second stimulus showed always a weaker response. Any combination of the stimuli confirmed that CYCAM1 is involved in all responses.To test whether the cyt elevation restricts SA accumulation, which becomes harmful if the mutant is exposed to SA-stimulating biotic and abiotic stress.A Figure B and SA-athogens ,68 and rt plants , similarignaling  and the ignaling -71. TherA. brassicae infection induced higher JA levels in cycam1 than in WT seedlings, while the levels in unchallenged WT and mutant seedlings is almost identical  and its AAL-Tox is well established for tomato cyt measurements, approximately 70% of the roots per seedling were dissected and incubated overnight in 150 μl of 7.5 μM coelenterazine  in the dark at 20°C in a 96 well plate . For cotyledon assays, the same protocol was used except that the root material was replaced by 3 leaves of the seedlings grown under the same conditions. For the leaf assay, ~ 1/32 part of a fully developed leaf (without middle rib) of 4 week-old plants grown in pots under LD conditions were used. Bioluminescence counts from roots or cotyledons/leaves were recorded as relative light units (RLU) with a microplate luminometer . The mutant screen was performed with the CWE from A. brassicae; the putative M2 mutants were rescued and transferred to pots containing garden soil and vermiculite at 9:1 (v/v) for further screening and validation. The mutant seedlings were grown in a temperature-controlled growth chamber under short day (SD) condition  for 4 weeks followed by LD condition in Aracon tubes. The seeds were harvested from individual M3 plants and again screened to obtain homozygote mutants.Aequorin based luminescence measurements were performed using 16-day old individual Md medium ,76. pMAQd medium . For cyt elevation measurements and combined cyt measurements and physiological studies.A Tox fraction from n et al.  with modcycam1 mutant were placed on MS medium  for SA; m/z 140.9 → 97.0  for D4-SA; m/z 209.1 → 59.0  for JA; m/z 213.1 → 56.0  for 9,10-D2-9,10-dihydrojasmonic acid; m/z 263.0 → 153.2  for ABA; m/z 269.0 → 159.2  for D6-ABA; m/z 322.2 → 130.1  for JA-Ile conjugate; m/z 328.2 → 136.1  for JA-13C6-Ile conjugate. Both Q1 and Q3 quadrupoles were maintained at unit resolution. Analyst 1.5 software (Applied Biosystems) was used for data acquisition and processing. Linearity in ionization efficiencies were verified by analyzing dilution series of standard mixtures. Phytohormones were quantified relative to the signal of their corresponding internal standard. For quantification of 12-oxophytodienoic acid, cis-OPDA, 9,10-D2-9,10-dihydro-JA was used as the internal standard applying an experimentally determined response factor of 1.100 mg of leaf material was frozen in liquid nitrogen and kept at -80˚C. After grinding with mortar and pestle, the leaf material was extracted with 1.2 ml of methanol containing 24 ng of 9,10-Dl et al.  using 13m/z 201.09 → 59.01 . Both Q1 and Q3 quadrupoles were maintained at unit resolution. Analyst 1.5 software  was used for data acquisition and processing. Linearity in ionization efficiencies was verified by analyzing dilution series of samples containing camalexin. A relative quantification of camalexin was performed by calculating peak area per mg of fresh weight.Samples were freeze-dried until constant weight and ground to a fine powder. Ten to fifty mg of freeze-dried and pulverised material was used for camalexin measurement by LC-MS analysis. Camalexin was analysed in the flow-through samples resulting from the extraction procedure for glucosinolate analysis (see below). In glucosinolate extraction, the raw extract was loaded onto DEAE Sephadex, and the resulting flow-through was collected in a 96 deepwell plate and directly analysed by LC-MS/MS. Chromatography was performed on an Agilent 1200 HPLC system . Separation was achieved on a Zorbax Eclipse XDB-C18 column . Formic acid (0.05%) in water and acetonitrile were employed as mobile phases A and B, respectively. The elution profile was: 0-0.5 min, 5% B; 0.5-1 min, 5-100% B in A; 1-2 min 100% B and 2.1-4. 5 min 5% B. The mobile phase flow rate was 0.8 ml/min. The column temperature was maintained at 25°C. An API 3200 tandem mass spectrometer  equipped with a Turbospray ion source was operated in positive ionization mode. The instrument parameters were optimized by infusion experiments. The ionspray voltage was maintained at 5500 V. The turbo gas temperature was set at 700°C. Nebulizing gas was set at 70 psi, curtain gas at 35 psi, heating gas at 70 psi and collision gas at 2 psi. Multiple reaction monitoring (MRM) was used to monitor analyte parent ion → product ion: −1). Detection was performed with a photodiode array detector and peaks were integrated at 229 nm. We used the following response factors: a-GLS 2.0, iGLS 0.5 [Samples were freeze-dried until constant weight and ground to a fine powder. Ten to fifty mg of freeze-dried and pulverised material per plant was used for GLS analysis. GLS were extracted with 1 ml of 80% methanol solution containing 0.05 mM intact 4-hydroxybenzyl GLS as internal standard and desulfated with arylsulfatase (Sigma-Aldrich) on a DEAE Sephadex A 25 column. The eluted desulfoglucosinolates were separated using high performance liquid chromatography  on a reversed phase column  with an water-acetonitrile gradient . ROS measurements were performed using the substrate carboxy-H2DFFDA (Molecular Probes) according to the manufacturer’s instructions . The plant material was incubated in 20 μM carboxy-H2DFFDA prepared in KRPG buffer for 30 min in the dark. The fluorescence intensity was quantified with a fluorescence microplate reader (TECAN Infinite 200) with an excitation at 485 nm and emission at 530 nm. The reaction mixture without the substrate and plant material served as control.Quantitative ROS measurement were performed using the Amplex Red hydrogenperoxide/peroxidase assay kit (Molecular Probes) according to the manufacturer’s instructions . cDNA was synthesised with the Omniscript cDNA synthesis kit (Qiagen) and 1 μg RNA. The oligonucleotide primers are given in Additional file  control . Real-tif Pfaffl . The rat2+]cyt: Cytosolic calcium; cycam1: Cytosolic calcium elevation mutant1; Chl: Chlorophyll; cis-12-OPDA: cis-12-oxo-phytodienoic acid; CWE: Cell wall extract; EMS: Ethyl methane sulfonate; EPM: Extract preparation from mycelia; EPS: Extract preparation from spores; GLS: Glucosinolates; iGLS: Indolic glucosinolates; JA: Jasmonic acid; JA-Ile: Jasmonyl-isoleucine; PA: Phsophatidic acid; PAMP: Pathogen-associated molecular pattern; ROS: Reactive oxygen species; SA: Salicylic acid; Tox: Toxin WT, wild-type.ABA: Abscisic acid; aGLS: Aliphatic glucosinolates; cyt elevation in Arabidopsis roots after treatment with A. brassicae PAMPs or toxin. Figure S2. Physical and chemical properties of CWE, EPM, EPS and toxin (Tox) from A. brassicae. Figure S3. Response of WT and cycam to CWE and EPM from Rhizoctonia solani , Phytophthora parasitica var. nicotianae , and the CWE from Agrobacterium tumefaciens (E). Figure S4. The cycam1-1 and cycam1-2 are more susceptible to Rhizoctonia solani and Phytophthora parasitica var. nicotianae infection. Figure S5. Photosynthetic parameters are impaired in cycam in response to different abiotic stress. Figure S6. Phytohormone regulated genes in WT and cycam seedlings. Table S1. Inhibition of [Ca2+]cyt elevation induced by the A. brassicae-derived CWE, EPM, EPS and Tox preparations by staurosporine in WT roots. Table S2. Primer list for RT-PCR. Dose dependent increase of [CaClick here for file


Aging is the summation of many subtle changes which result in altered cardiovascular function. Impaired endothelial function underlies several of these changes and precipitates plaque development in larger arteries. The endothelium transduces chemical and mechanical signals into changes in the cytoplasmic calcium concentration to control vascular function. However, studying endothelial calcium signaling in larger arteries in a physiological configuration is challenging because of the requirement to focus through the artery wall. Here, pressure- and agonist-sensitive endothelial calcium signaling was studied in pressurized carotid arteries from young (3-month-old) and aged (18-month-old) rats by imaging from within the artery using gradient index fluorescence microendoscopy. Endothelial sensitivity to acetylcholine increased with age. The number of cells exhibiting oscillatory calcium signals and the frequency of oscillations were unchanged with age. However, the latency of calcium responses was significantly increased with age. Acetylcholine-evoked endothelial calcium signals were suppressed by increased intraluminal pressure. However, pressure-dependent inhibition of calcium signaling was substantially reduced with age. While each of these changes will increase endothelial calcium signaling with increasing age, decreases in endothelial pressure sensitivity may manifest as a loss of functionality and responsiveness in aging. The endothelium is a single-cell-thick layer that lines the inner surface of all blood vessels and is exposed to numerous activators, such as blood-borne signaling molecules, electrical signals, and mechanical forces. The endothelium responds to these activators to control vascular function by releasing vasoactive agents such as nitric oxide (NO) and prostaglandins and by initiating endothelium-dependent smooth muscle hyperpolarization. With advancing age, the ability of the endothelium to control vascular tone is attenuated in animal models and in humans ,2,3,4. T2+ concentration ([Ca2+]i) is a major trigger for the agonist-induced production of NO i consist of an initial transient increase in intracellular Ca2+, due to inositol trisphosphate (IP3)-mediated release from the ER, followed by a sustained elevation maintained by Ca2+ influx from the extracellular space i signaling responses to ACh using gradient index (GRIN) fluorescence endomicroscopy propane-sulfonic acid) (2.0), NaH2PO4 (1.2), glucose (5.0), EDTA (ethylenediaminetetraacetic acid) (0.02), MgCl2 (1.17), and CaCl2 (2.0).All animal care and experimental procedures were carried out with the approval of the University of Strathclyde Animal Welfare and Ethical Review Body and authorized under UK Home Ofﬁce regulations . Young (3-month-old) and aged (18-month-old) male Sprague-Dawley rats (250-350 g) were killed by overdose of pentobarbital sodium . Subsequently, carotid arteries were quickly removed and placed in chilled physiological saline solution  composed of  and Pluronic F127 . Once the Ca2+ indicator was introduced to the lumen, flow was stopped, and the endothelium allowed to load for 30 min. The final (working) concentrations of Pluronic F127 and DMSO were 0.04 and 0.96%, respectively. Following loading, excess dye was then flushed from the lumen , the distal cannula was removed, and the artery was mounted onto a side-viewing GRIN microendoscopic-imaging probe and repressurized. Throughout the loading procedure, the artery was continuously superfused with PSS that was warmed to 37°C before entering the bath.Caopy Fig. , as prev2+ responses were recorded for 60 s after addition of ACh to the bath solution. Noncumulative concentration response experiments were carried out in arteries pressurized to 60 mm Hg; 60 mm Hg was selected because it is an exceptionally widely used pressure in myograph studies permitting comparison of the present results to the findings of others. During all imaging experiments, superfusion was halted to avoid movement artifacts, and the reported concentrations of ACh are final bath concentrations. Note that the ACh concentration when applied to the outside of the artery is 1,000 fold higher than required when ACh has free access to the endothelium  to wash ACh from the bath, and arteries were allowed to reequilibrate for 20 min before the next stimulation. Importantly, in these experiments, the response of each individual endothelial cell was matched across each ACh concentration. The response of individual cells was normalized to the maximal averaged response. Endothelial Ca2+ signaling was also assessed at various intraluminal pressures. In pressure experiments, arteries were first stimulated with 100 µM ACh at 60 mm Hg, as this concentration was found to activate the majority of cells in the field of view (FOV). Following washout of ACh, intraluminal pressure was increased, from 60 to 110 mm Hg and subsequently from 110 to 160 mm Hg. Arteries were allowed to equilibrate at each experimental pressure for 20 min before stimulation. In the pressure change experiments, matching of individual cells was not possible because of cell movement that occurred as artery diameter changed. The response of individual cells was normalized to the maximal averaged response at 60 mm Hg. In all experiments, images were acquired at 5 Hz using µManager software i in response to ACh  resulted in both a graded increase in the number of cells activated and the amplitude of response in each cell , and sustained increases with repetitive oscillations occurred at higher ACh concentrations  rats was significantly more sensitive than that of young  rats (p < 0.05).Endothelial cells of intact and pressurized rat carotid arteries from young animals exhibit heterogeneous concentration-dependent rises in [CaACh Fig.  38]. Th. Th2+]i ous Fig. . Increasell Fig. , 2b. Notion Fig. , 2c. To ion Fig. , circles2+ response evolved from a transient Ca2+ increase that quickly returned to baseline, to a sustained increase with multiple oscillations  sufficient to activate the majority of cells across the FOV. Temporal metrics were calculated from discrete derivate Ca2+ signals  nor the frequency of oscillations  differed between young and aged rats. However, the temporal spread of the Ca2+ responses  was examined in individual arteries from aged animals, subject to stepwise pressure changes  and compared to the pressure response of young animals. Like the response in young animals  rats and compared to that in young (3-month-old) rats. The results show that while the endothelium was more sensitive to ACh, pressure-dependent modulation of agonist signaling is attenuated with age. These alterations both contribute, paradoxically, to the increase in endothelial Ca2+ signaling with age.Changes in arterial structure, associated with aging, increase tissue stress and degrade vascular function. Among the cellular components of the vascular wall, endothelial cells may be the most predisposed to age-related structural alterations. Endothelial Caagonists . Howeveractivation of the endothelium. The response of the endothelium to pressure is less well understood, but differs significantly from that of shear stress. Indeed, a decrease in activity occurs as pressure is increased i is increased  at IP3R clusters following the release of the ion because of the restricted diffusional space. The increased local [Ca2+]i at IP3R reduces the entropic force driving Ca2+ release and so decreases total Ca2+ liberated from the ER i is generally considered essential for the agonist-induced production of NO i during muscarinic activation has been proposed to compensate for age-related reductions in NO bioavailability [Caon of NO ,17,18. Eon of NO ,65,66 anon of NO . Given tlability ,69,70 bylability .2, thromboxane A2[2+ signals by increased pressure in young animals is attenuated with age. As a result of the decreased pressure sensitivity, Ca2+ signaling would be maintained as pressure is increased and may help minimize the physiological consequences of a decrease in eNOS activity with age [2+ signaling is paradoxically increased and may serve to offset other decreases in endothelial function, such as decreased eNOS activity, and maintain vascular contractile function.Alterations in the mechanical properties of blood vessels and altered production of endothelium-derived vasoactive factors such as endothelin , prostagboxane A2, and NO boxane A2,6,7,8 arwith age ,7 Thus, The authors have no conflicts of interest to disclose.


In pathological conditions such as ischemia reperfusion injury, Ca2+ overload into mitochondria induces mitochondrial permeability transition (MPT), a critical step for cell death. Because inhibition of MPT is a promising approach to protecting cells and organs, it is important for drug discovery to identify novel chemicals or mechanisms to inhibit MPT. Here we report upon a small-molecule compound DS44170716 that inhibits Ca2+-induced MPT in rat liver isolated mitochondria. DS44170716 protects human liver HepG2 cells from Ca2+-induced death with a level of protection similar to cyclosporin A (CsA). The inhibitory mechanism of DS44170716 against MPT is independent on PPIF, a target of CsA. DS44170716 blocks Ca2+ flux into the mitochondria by decreasing mitochondrial membrane potential, while potently inhibiting mitochondrial complex III activities and weakly inhibiting complex IV and V activities. Similarly, complex III inhibitor antimycin A, complex IV inhibitor KCN or complex V inhibitor oligomycin inhibits Ca2+ uptake of isolated mitochondria. These results show that DS44170716 is a novel class inhibitor of MPT by blocking of mitochondrial complexes and Ca2+-overload into mitochondria.Mitochondria are involved in a variety of physiological and pathological processes. Ca Mitochondrial complexes I to IV produce a proton gradient across the inner membrane of mitochondria, and complex V converts ADP to ATP by using energy of the membrane potential. In addition to their role as a cellular energy source, mitochondria maintain cellular Ca2+ homeostasis by uptake of Ca2+ in response to cytosolic Ca2+ increase3. In a pathological condition, a massive amount of Ca2+ enters mitochondria and induces mitochondrial permeability transition (MPT)4. The MPT causes disruption of the mitochondrial membrane and releases cytochrome c, which triggers several necrotic or apoptotic cell death cascades.Mitochondria generate cellular energy by oxidative phosphorylation2+ overload is involved in various pathological events, including ischemia-reperfusion injury5. Immunosuppressant cyclosporin A (CsA) has been shown to inhibit MPT and protect against cellular damage in several tissues7. NIM-811, a non-immunosuppressive analog of CsA, also protects against ischemia-reperfusion injury11, showing that the mitochondrial action of CsA is critical for the protective effect. Sanglifehrin A and antamanide also inhibit MPT and cell death by inhibiting the same target, peptidyl-prolyl cis-trans isomerase F (PPIF)13. PPIF-deficient mice show protective effects in a variety of pathological models in addition to that of reperfusion injury18. These results suggest that the development of an MPT inhibitor would be a promising approach to protecting cells and tissues.Mitochondrial Ca19. Because atractyloside or bongkrekic acid, inhibitor of adenine nucleotide translocator (ANT), affects MPT, ANT was thought to be required for MPT20. However, mitochondria from ANT-deficient mice reveal that ANT is not essential for MPT induction but is involved in Ca2+ sensitivity of MPT21. Ro68-3400 was identified by a screening of small molecule compounds, and it potently inhibits mitochondrial swelling. The compound was demonstrated to bind to voltage-dependent anion channel (VDAC), suggesting VDAC is a potential mediator of MPT22. However, VDAC was shown to be dispensable to MPT by a loss of function study of VDAC23. PPIF, the target of CsA in the mitochondrial matrix, is the most well-known mediator of MPT. Actually, mitochondria from PPIF-deficient mice have been demonstrated to show resistance to swelling induced by Ca2+ 25. In addition, recent studies identified several inhibitors of MPT29. These compounds seem to be valuable chemical biological tools to understanding mechanism of MPT, but molecular mechanisms of their inhibitory actions are completely unknown.Despite intensive studies in biochemistry and molecular biology, the mechanism of MPT remains unsolved2+-induced mitochondrial swelling, and found its action to be independent of PPIF. The compound protected human liver cultured cells from Ca2+ ionophore-induced death with a level of protection similar to CsA. Further analysis revealed that DS44170716 potently blocks Ca2+ overload into mitochondria by inhibiting mitochondrial complex III, IV and V. The study shows that the blockade of Ca2+ influx by inhibition of mitochondrial complex activities is a novel mechanism for protection against Ca2+-induced cell death.In the present study, we screened small molecule compounds for MPT inhibitors by using isolated mitochondria. We identified DS44170716 as showing a significant protective effect against Ca29. In the present study, freshly-isolated mitochondria from rat liver were used for the swelling assay. Application of 100 μM Ca2+ to mitochondrial solution resulted in a temporal decrease in absorbance at 540 nm. We identified a compound DS44170716 that dose-dependently inhibited decrease of absorbance in a range of 1 μM to 100 μM  within the supernatant of the cell culture. The CsA treatment protected cells from Ca2+-induced cell death  for strong induction of mitochondrial swelling  to evaluate binding activity of the compounds to PPIF. After incubation of radio-labeled CsA with PPIF, the binding levels were quantified by using SPA beads following immunoprecipitation with an anti-PPIF antibody. Binding of radio-labeled CsA to PPIF was competitively inhibited by preincubation of non-labeled CsA in a dose-dependent manner . Then we used fresh, isolated mitochondria from pig heart. The mitochondria were incubated with 100 μM Ca2+, and then intramitochondrial Ca2+ levels were detected. We detected a significant increase of Ca2+ levels in the mitochondria  also blocked accumulation of mitochondrial Ca2+ in a dose-dependent manner  showed a smaller infarct than that seen in those receiving the placebo. On the other hand, the phase 3 clinical study using 970 patients revealed that CsA showed no significant effect on the reperfusion injury after PCI44. As discussed in the reports, an inhibitor specific to MPT may be important for clinical efficacy because CsA has nonmitochondrial effects including potent immunosuppressive activity. Therefore, a PPIF-independent target is one of the next candidates for development of MPT inhibitors29.In drug development research, the regulation of MPT is thought to be a promising target for tissue protection. CsA and its non-immunosuppressive analogs have been shown to protect from reperfusion injury in several animal studies51. In addition, a human genetic study showed that a mutation of MICU1, the gene encoding a regulatory subunit of the uniporter, causes an increase of mitochondrial Ca2+ influx and promotes the development of brain and muscle disorders52. These studies suggest that blocking the mitochondrial Ca2+ entry is a promising approach to protecting tissue from injury. The present study identifies a novel MPT inhibitor that blocks mitochondrial Ca2+ uptake by interfering with the enzyme activities of mitochondrial complexes. Because DS44170716 directly inhibits respiratory chains, the compound may have safety problem in the respect of drug development. For the purpose, a direct inhibitor of the MCU seems to be hopeful as a next mitochondria-targeting drug. Recently, we found a cell-permeable blocker of the uniporter by using a novel screening method (in preparation for publication). Blockade of Ca2+ overload is an emerging strategy for drug development seeking to satisfy the unmet medical needs of mitochondrial diseases.Recent molecular biological studies have identified components of the mitochondrial calcium uniporter54. The following were also purchased: fresh pig hearts from Tokyo Shibaura Zouki Co., Ltd. ; pIRES-puro vector from Clontech Laboratories, Inc. ; HEK293A cells, pcDNA3.1, Hanks Balanced Salt Solution, coelenterazine h and mitochondria isolation kit from Thermo Fisher Scientific Inc. ; MitoTox OXPHOS Complex III Activity Kit, Complex IV Human Enzyme Activity Microplate Assay Kit and MitoTox Complex V OXPHOS Activity Microplate Assay from Abcam Plc ; fetal bovine serum and ruthenium red, rotenone, antimycin A, KCN, oligomycin, FCCP, genipin and cyclosporin A from Sigma-Aldrich, Inc. ; JC-10 from Enzo Life Sciences, Inc. ; 2-Thenoyltrifluoroacetone (TTFA) from Caymen Chemical ; DS44170716 from specs . NIM-811 was synthesized as described previously55.Nine-week-old male Wistar rats were purchased from Japan SLC . All experimental procedures were performed in accordance with the in-house guidelines of the Institutional Animal Care and Use Committee of Daiichi-Sankyo, which is an accredited organization by Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The experimental protocols for the present study were approved by the in-house committee (protocol application number A1400451). Every effort was made to minimize animal suffering and to reduce the number of animals employed. All animal studies were also conducted in accordance with the ARRIVE guidelinesg, 10 min), and mitochondria were then spinned down at a higher speed . All procedures were carried out at 4 °C.Mitochondria were isolated from rat livers or pig hearts through sequential centrifugation by using a mitochondria isolation kit for tissue . To obtain mitochondria, tissues were disrupted with a glass Teflon homogenizer. Nuclei and plasma membrane fractions were separated first by mild centrifugation . Five minutes after application of DS44170716, 100 μM CaCl2 was added and OD (540 nm) was measured per minute for 30 min by using a SpectraMax Plus (Molecular Devices). In order to test PPIF-dependency, NIM-811, RuR or DS44170716 was applied in the mitochondrial suspension with 10 μM CsA, and then 500 μM CaCl2 was applied.Swelling of mitochondria was detected as described previouslyCyclosporin A or DS44170716 was incubated with 100 nM human PPIF protein  in binding buffer  for 3 h at room temperature. Then, 100 nM tritium-labeled cyclosporin A  was added and incubated for 1 h. To immunoprecipitate PPIF protein, 20 ng of anti-PPIF antibody  was added and incubated for 1 h. SPA beads  were added and incubated for 20 min, then the radioactivity levels were measured with a TopCount® .2  to the solution, the mitochondria were collected by centrifugation  at 4 °C. The pellets were resuspended in the swelling buffer containing 1 μM ruthenium red. After collection of the mitochondria by centrifugation , the pellets were dried and dissolved by 40 μL of sulfuric acid at 95 °C. The solution was then diluted by water, and Ca2+ concentration of the solution was measured by atomic absorbance spectrometer .Mitochondria were isolated by using the mitochondria isolation kit and then dissolved in the swelling buffer. Thirty minutes after application of CaClPig heart mitochondria were prepared as described above and suspended in the swelling buffer containing 1 μM JC-10. The protein concentration of the solution was 5 mg/mL. Five minutes after incubation of the mitochondria with DS44170716 at room temperature, fluorescence intensities were measured by a FlexStation® 3  using the green channel (excitation/emission wavelength: 485 nm/538 nm) or the red channel (excitation/emission wavelength: 485 nm/612 nm).2+ levels in intact cells, HEK293A cells were stably transfected with pIRES-puro vector expressing mitochondria-targeted aequorin32. One day after plating on a 15-cm dish (8 × 106 cells/dish), the cells were harvested and incubated with 2.5 μM coelenterazine h in aequorin assay buffer  for 2 hours at room temperature. The cells (8.1 × 104 cells/well) were then treated with DS44170716 for 20 min in a 96-well plate at room temperature. For induction of intracellular Ca2+, the cells were treated with 10% fetal bovine serum, and luminescence levels were measured by using a Centro LB960 luminometer (Berthold Technologies USA).For dynamic measurements of mitochondrial Ca4 cells/well) in a 96-well dish with DMEM , cells were treated with medium  containing 10 μM A23187 and drugs for 3 hours. Then, supernatant was collected and LDH activities were measured by a Cytotoxicity Detection Kit (LDH) .One day after plating of HepG2 cells (5 × 10http://www.gen-info.osaka-u.ac.jp/MEPHAS/). The data presented in the Figures are mean with SEM of 3 or 4 technical replicates as described in the legends. Similar results were obtained in at least 3 independent biological replicates.For multiple comparisons of experimental data, Dunnett’s test was performed by using web-based statistical analysis tools (Supplemental Information


Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues , which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins . We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.Visinin-like protein 3 (VILIP-3) belongs to a family of Ca Two-dimensional 1H-15N HSQC spectra of VILIP-3 were recorded at 30°C as described previously /[Protein]) was measured as a function of free Ca2+ concentration using flow-dialysis Ca2+ binding experiments  were monitored by ITC  and floweriments  as descr2+ with Hill coefficient (α) of 1.8 and apparent dissociation constant (KD) equal to 0.52 μM. The VILIP-3 mutants (E26A and F64A) each bound to Ca2+ with higher apparent affinity compared to wild type (2+-free VILIP-3. These mutants increase the Ca2+-binding affinity by destabilizing the Ca2+-free VILIP-3 structure (with sequestered myristoyl group) more so than the Ca2+-bound state (extruded myristate), which makes the free energy of Ca2+ binding more negative and hence more favorable. By contrast, the corresponding mutants in recoverin (E27A and Y65A) did not affect Ca2+ binding affinity  is lower than the unfolding temperature of myristoylated VILIP-3 (Tm = 57°C), consistent with a stabilization caused by sequestration of the covalently attached myristoyl group inside Ca2+-free VILIP-3. The myristoylated VILIP-3 mutants (E26A and F64A) exhibited a detectably lower folding stability  experiments were performed on VILIP-3 to measure the effect of myristoylation on protein folding stability. Representative DSC scans of wild type VILIP-3 are shown in 2+ saturated myristoylated VILIP-3, the protein started to aggregate at around 42°C and the precise unfolding temperature could not be accurately measured by DSC , α2 (residues 46–56), α3 (residues 62–72), α4 (residues 82–91), α5 (residues 101–108), α6 (residues 118–129), α7 (residues 146–156), α8 (residues 166–176), α9 (residues 179–187), β1 (residues 42–44), β2 (residues 79–81), β3 (residues 115–117) and β4 (residues 163–165)  . The cov163–165)  that pla163–165)  is uniquresidues 8–129, α7163–165) . Each EFecoverin . The int VILIP-3  is quiteresidues 8–129, α72+-bound myristoylated VILIP-3 -edited and (13C/F3)-filtered NOESY-HSQC) performed on Ca2+-free VILIP-3 samples that contained a 13C-labeled myristoyl group (13C-labeled fatty acyl chain. Representative Nuclear Overhauser effect (NOE) dipolar interactions are shown for the C14 methyl of the myristoyl group  and both helices of EF2 . The spectrum that probes the C12-position of the myristoyl moiety  to myristoylated VILIP-3 in the sub-micromolar range . A Hill coefficient of 1.8 is consistent with 3 Ca2+ binding sites in VILIP-3 having positive cooperativity, which resembles the cooperative Ca2+ binding observed for myristoylated recoverin [2+-free myristoylated VILIP-3 has a higher unfolding temperature than the Ca2+-free unmyristoylated protein, consistent with protein stabilization caused by the covalently attached myristoyl group. The fatty acyl group is sequestered inside a unique hydrophobic core of Ca2+-free VILIP-3 that involves myristate contacts to E26, Y64 and V68  and hippocampal synaptic plasticity in learning and memory. The Ca2+-dependent regulation of sAHP channels mediated by hippocalcin, therefore, might be similar to Ca2+-dependent regulation of voltage-gated Ca2+ channels mediated by CaM [2+-free and Ca2+-bound forms of CaM each bind to separate sites on the CaV1.3 channel [2+-bound CaM binds to an N-terminal site , which is responsible for inhibiting channel activity [2+-free and Ca2+-bound forms of VILIP-3 both bind to distinct regulatory sites on channel targets.The physiological target proteins that bind to VILIP-3 are currently not known. Hippocalcin, a close homolog of VILIP-3 (94% identity), binds and regulates Cad memory . The verd by CaM ,49. The  channel . By contactivity ,51. Hipp


This study explored the effect of longer duration nsPEF on [Ca2+]i. Single 150, 200, or 400 ns pulses at 3.1 kV/cm evoked rapid increases in [Ca2+]i, the magnitude of which increased linearly with pulse width and electric field amplitude. Recovery of [Ca2+]i to prestimulus levels was faster for 150 ns exposures. Regardless of pulse width, no rise in [Ca2+]i occurred in the absence of extracellular Ca2+, indicating that the source of Ca2+ was from outside the cell. Ca2+ responses evoked by a 150 ns pulse were inhibited to varying degrees by ω-agatoxin IVA, ω-conotoxin GVIA, nitrendipine or nimodipine, antagonists of P/Q-, N-, and L-type VGCCs, respectively, and by 67% when all four types of VGCCs were blocked simultaneously. The remaining Ca2+ influx insensitive to VGCC inhibitors was attributed to plasma membrane nanoporation, which comprised the E-field sensitive component of the response. Both pathways of Ca2+ entry were inhibited by 200 μM Cd2+. These results demonstrate that, in excitable chromaffin cells, single 150–400 ns pulses increased the permeability of the plasma membrane to Ca2+ in addition to causing Ca2+ influx via VGCCs. Exposing adrenal chromaffin cells to 5 ns electric pulses (nsPEF) causes a rapid rise in intracellular Ca It is not surprising, therefore, that a great deal of attention has been directed at developing techniques to manipulate intracellular Ca2+ levels ([Ca2+]i) in new ways. One such technique is the application of nanosecond electric pulses (nsPEFs) to biological cells.Calcium  that form in the lipid bilayer [2+ to enter cells. The second pathway involves Ca2+ entry mediated by activation of ion channel proteins [Exposing cells to nsPEF causes an increase in [Caeticulum –5. Most ytoplasm . In addism [2+]i . On the  bilayer –12. Thatproteins , 14.2+]i in particular cell types in conjunction with an understanding of the basis for differences in how various cell types undergo changes in [Ca2+]i in response to nsPEFs is essential for developing nsPEF-based applications. Our specific goal has been to work toward the potential for nsPEF to serve as a novel electrostimulation modality for altering neural cell excitability. To this end we have been exploring the response of isolated adrenal chromaffin cells, a well-established nontransformed model of neural-type cells, to nsPEF in the low nanosecond regime (less than 10 ns in duration). In these cells, depolarizing stimuli cause Ca2+ influx via voltage-gated Ca2+ channels (VGCCs), which in turn triggers release of catecholamines by exocytosis [2+ influx that occurs solely through VGCCs [2+ from internal stores [2+ across the plasma membrane. The latter was established both in Ca2+ imaging experiments showing the absence of a rise in [Ca2+]i when VGCCs were blocked i was used to investigate Ca2+ responses in chromaffin cells exposed to single, longer duration nsPEF, specifically pulses having widths of 150, 200, or 400 ns. Such longer duration pulses may well have more profound effects on plasma membrane permeability which, from a physiological standpoint, would have important implications regarding chromaffin cell function and the potential use of such pulses for neuromodulation.Continuing to explore the potential use of nsPEF for modulating neural cell excitability, we describe here the results of studies in which fluorescence imaging of i that was maximal after 1 to 2 s. Over the course of a 50 s monitoring period, [Ca2+]i recovered to baseline in half the cells exposed to a 150 ns pulse but was more sustained and did not reach baseline levels in the remaining cells. A similar pattern of a more sustained rise in [Ca2+]i elicited by nsPEF was also observed in cells exposed to a single 200 or 400 ns pulse. It is important note that the recovery of [Ca2+]i to baseline in cells exposed to nsPEF, even pulses of a 5 ns duration, is much slower than in cells stimulated with the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium i was intracellular or extracellular, cells were exposed to a 150, 200, or 400 ns pulse at 3.1 kV/cm in Ca2+-free BSS that contained EGTA. As shown in 2+ abolished the response of the cells to nsPEF exposure for the three pulse durations tested. Thus, regardless of pulse duration, the mechanism by which 150–400 nsPEF exposure caused an increase in [Ca2+]i in chromaffin cells at an E-field of 3.1 kV/cm, which is just above threshold for evoking a response , and Ca2+ mobilization is not evoked in chromaffin cells exposed to 5 ns pulses, the ability of longer duration pulses to cause an intracellular permeabilizing effect is unlikely. Perhaps increasing the E-field amplitude of 150–400 ns pulses would lead to detectable Ca2+ mobilization from internal stores, as has been shown for cell exposures to 5 ns pulses i by 9%, although the effect did not reach statistical significance. These results are comparable to those observed in cells exposed to 5 ns pulses i was reduced by the VGCC cocktail, 48% (p < 0.01), reflected primarily the combined inhibition of P/Q- and N-type channels. Thus, complete inhibition by the VGCC cocktail on Ca2+ influx evoked by a 150 ns pulse, as found for a 5 ns pulse, was not observed due to the lack of an inhibitory effect of dihydropyridines on L-type channels.The selective P/Q-type channel antagonist s pulses . Howeverns pulse . Nitrend2+ influx. In chromaffin cells, L-type channels comprise both Cav1.2 and Cav1.3 isoforms that are sensitive to dihydropyridines i in response to a 150 ns pulse when Cd2+ was present, indicating that this divalent ion not only blocked VGCCs but also Ca2+- permeable nanopores that were presumably formed in the plasma membrane. These results are interesting from two standpoints. First, Roth et al. (2013) reported that in mouse primary hippocampal neurons exposed to nsPEF, Cd2+ failed to prevent Ca2+ uptake into the cells due to plasma membrane permeabilization [2+ [3+ was a potent blocker of membrane conductance [2+ is known to bind to the same high affinity binding site for Ca2+ inside the VGCC pore and thus block Ca2+ entry [2+ can effectively compete with Ca2+ that is present at a tenfold higher concentration to block Ca2+ entry via a putative lipid nanopore. Future studies addressing this issue are needed to understand the basis for differences in plasma membrane conductance properties evoked by nsPEF in various cell types.Cdlization . Pakhomotion [2+ . Their sductance . Second,2+ entry , 34, it + and not Ca2+ [2+ influx would now allow YO-PRO-1 to be taken up by the cells. To address this question, 150 ns pulses were delivered to the cells in BSS containing 2 μM YO-PRO-1. In cells exposed to a single pulse applied at 3.1 kV/m, YO-PRO-1 uptake was never observed (results not shown). Thus, even though the plasma membrane was now permeable to Ca2+, chromaffin cells were still resistant to YO-PRO-1 uptake, indicating that Ca2+ was entering the cells via nanopores that were still too small to permit YO-PRO-1 passage. Moreover, as shown in E-field was increased to 9.7 kV/cm, 20 pulses caused YO-PRO-1 uptake that was accompanied by significant cell swelling (The fluorescent dye YO-PRO-1 has been used as an indicator of plasma membrane permeabilization to nsPEF . Interesnot Ca2+ . In the swelling . Thus, f2+. In isolated adrenal chromaffin cells exposed to 5 ns pulses, the plasma membrane does not become permeable to Ca2+ and influx of Ca2+ occurs only via VGCCs. However, increasing pulse duration to 150 ns causes significant Ca2+ influx via plasma membrane nanopores in addition to Ca2+ influx through VGCCs. Nevertheless, regardless of whether the enhanced permeabilizing effect is the result of larger pores or a different structure of the pores due to the longer application of the electric field, the cells still did not take up YO-PRO-1 unless they were exposed to a large number of nsPEFs at a high electric field intensity that causes significant cell swelling. Thus, pulse duration and amplitude, which do not affect VGCC-mediated Ca2+ influx, that is, an all-or-none response, appear to selectively modify membrane conductance to ions in this cell type in a manner that still limits YO-PRO-1 uptake. Taken together, our data suggest that neurosecretion could be fine-tuned by modulating the Ca2+ response through manipulation of nsPEF amplitude and duration. Studies examining these possibilities are underway.A hallmark feature of cells exposed to nsPEFs is the formation of nanometer-size pores in the plasma membrane that, in many cell types, renders the membrane permeable to Ca


Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is a multi-functional protein that plays critical role in processes including transcription, transcription-coupled DNA repair, pre-mRNA splicing, homologous recombination and mRNA export. Microarray analysis on gene expression in XAB2 knockdown cells reveals that many genes with significant change in expression function in mitotic cell cycle regulation. Fluorescence-activated cell scanner analysis confirmed XAB2 depletion led to cell arrest in G2/M phase, mostly at prophase or prometaphase. Live cell imaging further disclosed that XAB2 knockdown induced severe mitotic defects including chromosome misalignment and defects in segregation, leading to mitotic arrest, mitotic catastrophe and subsequent cell death. Among top genes down-regulated by XAB2 depletion is mitotic motor protein centrosome-associated protein E (CENPE). Knockdown CENPE showed similar phenotypes to loss of XAB2, but CENPE knockdown followed by XAB2 depletion did not further enhance cell cycle arrest. Luciferase assay on CENPE promoter showed that overexpression of XAB2 increased luciferase activity, whereas XAB2 depletion resulted in striking reduction of luciferase activity. Further mapping revealed a region in CENPE promoter that is required for the transcriptional regulation by XAB2. Moreover, ChIP assay showed that XAB2 interacted with CENPE promoter. Together, these results support a novel function of XAB2 in mitotic cell cycle regulation, which is partially mediated by transcription regulation on CENPE. The XAB2 protein contains 15 tetratricopeptide repeat motifs involved in protein–protein interactions and the assembly of multiprotein complexes. It has many orthologues, such as SYF1 in Saccharomyces cerevisiae,3 ATH55 in rat4 and Fandango in Drosophila.5Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is a highly conserved gene, which was originally identified in human as a protein interacting with XPA using yeast two-hybrid system.6 Further studies showed that XAB2 might bind to hyperphosphorylated form of RNA polymerase II in a UV- and CSA/CSB-dependent manner7 and contribute to transcription elongation.6 Furthermore, XAB2 is implicated as a component of Prp19/XAB2 complex6  or Prp19/CDC5L-related complex8 required for pre-mRNA splicing. During splicing, Prp19 complex is recruited to the spliceosome and crucial for spliceosome activation by stabilizing U5 and U6 with the spliceosome after U4 dissociation.10 In Hela cells, Prp19/XAB2 complex was reported to associate with RNA rather than DNA, and knockdown of XAB2 would inhibit Bcl-x pre-mRNA splicing.6 Similarly, SYF1, homolog of XAB2 in yeast, is also identified as a factor involved in pre-mRNA splicing,3 and a novel transcription elongation factor by linking Prp19 complex with RNA polymerase II through its C terminal domain.11It was reported that XAB2 could interact with Cockayne syndrome group A and B (CSA and CSB) proteins as well as RNA polymerase II, and downregulation of XAB2 using anti-XAB2 antibody or siRNA both specifically inhibited normal RNA synthesis and the recovery of RNA synthesis after UV irradiation, indicating that XAB2 is involved in transcription and transcription-coupled DNA repair.12 It was previously demonstrated that XAB2 could interact with retinoic acid receptor A and histone deacetylase 3, and XAB2 deficiency increased all-trans retinoic acid (ATRA)-induced cellular differentiation in ATRA-sensitive and -resistant cancer cells. These results suggest that XAB2 may display an inhibitory effect in ATRA-induced cellular differentiation.13 Furthermore, XAB2 may play a role in tumorigenesis. XAB2 expression is significantly downregulated in primary gastric cancer,14 triple-negative breast cancer15 and a long-term survivor of an atypical teratoid/rhabdoid tumour,16 while it is upregulated in ovarian cancer17 and sarcoma18 using Oncomine database (www.oncomine.org). The XAB2 tagSNPs (rs794078 and rs4134816) are dramatically correlated with the risk of non-small cell lung cancer in Chinese population.19 In addition, XAB2 levels are strikingly reduced in aged hematopoietic stem cells, suggesting a role in ageing.20 Recently, we showed that XAB2 presented in the ribonucleo-protein of a consensus element found in naturally intronless mRNAs that can promote mRNA export, and disruption of XAB2 led to nuclear retention of the intronless mRNAs, suggesting a role of XAB2 in naturally intronless mRNA export.21 Most recently, XAB2 has been reported to regulate the end resection step during homologous recombination repair of chromosomal double-strand breaks.22 Thus, XAB2 is an important multifunctional protein.XAB2 is also essential for early mouse embryogenesis, because knockdown of XAB2 in mice has been shown to lead to embryonic lethality.Despite the critical functions of XAB2 in gene expression, the downstream target genes and the subsequent physiological roles in human cells are still unknown. Here, we perform microarray analysis on Hela cells transfected with XAB2 shRNA, revealing that XAB2 modulates the expression of a significant number of genes, most highly involved in cell cycle and mitotic progression. We further show that XAB2 knockdown causes chromosome misalignment and missegregation, abnormal nuclear structure, spindle pole, and microtubule organization, leading to mitotic arrest, mitotic catastrophe and subsequent cell death. Mechanistic analysis reveals that the phenotype observed in XAB2 depletion cells is mediated by a kinesin-like motor protein called centrosome-associated protein E (CENPE), the transcription of which is regulated by XAB2. Taken together, our data indicate that XAB2 regulates mitotic progression by transcription control of CENPE.6 splicing6 and mRNA export.21 To identify target genes regulated by XAB2, gene expression of XAB2 depleted HeLa cells was analysed by microarray. This analysis revealed that a total of 690 genes showed significant change in expression . Among them, 216 genes were up-regulated and 474 genes were down-regulated  analysis was performed on Hela cells transfected with XAB2 shRNA. Strikingly, XAB2 knockdown resulted in a significant increase of cells arrested in G2/M phase . The percentage of cells showing mitotic arrest or mitotic delay (mitosis duration >90 min) was raised dramatically from 4.9 to 81.2% (P<0.001). Furthermore, cells undergoing mitotic cell death were significantly increased after XAB2 knockdown from 3.9 to 65.8% (P<0.001). To further test whether XAB2 knockdown induced cell death after mitotic catastrophe, FACS analysis was performed after annexin V and propidium iodide (PI) staining. As shown in To better investigate the function of XAB2 on mitotic progression, we depleted XAB2 in HeLa cells stably expressing GFP-H2B and tracked mitosis using live cell imaging. As shown in to 81.2% , P<0.001to 65.8% , P<0.001Because mitosis requires a tight control of chromosome movement, we next performed DAPI staining to determine whether XAB2 affects chromosome alignment and segregation. This analysis revealed severe chromosome misalignment at equatorial plate and internuclear DNA bridges .Consistent with previous observation, most of the mitotic cells showed mitotic arrest, mitotic delay or segregation defect. Specifically, mitotic arrest in prophase or prometaphase with defects in chromosome alignment was observed in 43.1% of mitotic cells after XAB2 depletion compared to 2.6% in control, which did not divide and eventually led to cell death . Mitotic24 As shown in γ-H2A.X) was significantly increased in XAB2 knockdown cells by both immunofluorescence staining and western blot analysis. Together, these data suggested that XAB2 was critical for microtubule and spindle organization as well as genome stability.We next sought to further examine how mitosis is disturbed in XAB2 knockdown cells. First, spindle pole was checked in XAB2 depleted cells since correct spindle formation is required for chromosome alignment and segregation. Strikingly, single or multiple spindle poles was observed in ~30 and ~8% of prophase/prometaphase cells after XAB2 knockdown using either tubulin or Aurora A staining . Second,P<0.001). Meanwhile, CENPE depletion also induced chromosome misalignment and prophase/prometaphase arrest (78.1% in knockdown cells versus 17.2% in control), similar to the phenotype observed in XAB2 deficiency cells , including metaphase delay, metaphase alignment problems and cell deaths.25To gain deep insight into the impact of XAB2 deficiency, we performed microarray analysis on gene expression after XAB2 depletion in HeLa cells. Surprisingly, this analysis revealed that the expression of many genes functioning in cell cycle or mitosis regulation was changed significantly. We further showed that XAB2 deficiency induced chromosome misalignment and missegregation, abnormal nuclear structure, spindle pole, and microtubule organization, resulting in mitotic arrest, mitotic catastrophe and subsequent cell death, indicating a critical role of XAB2 in the regulation of mitotic cell cycle. Our results are consistent with phenotypes of XAB2 knockdown documented in the MITOCHECK database  has been reported to bind to RNA but not DNA in vitro and XAB2 contains 15 tetratricopeptide repeat motifs involved in protein–protein interactions but without DNA-binding domains, it is very likely that XAB2 was recruited to the promoter of CENPE by other proteins.We further showed that XAB2 binds to the promoter of CENPE and regulates its expression using ChIP and luciferase assay. Overexpression of XAB2 led to higher luciferase activity whereas XAB2 depletion resulted in striking decrease of luciferase activity. Previous report showed that XAB2 interacts with RNA polymerase II and plays a role in transcription, mostly by modulating transcription elongation.However, in the CENPE rescue experiment, we observed no significant restoration of cell cycle arrest when CENPE was re-expressed after XAB2 depletion (data not shown). Intriguingly, re-expression of CENPE after its own knockdown in Hela cells could not reverse cell cycle arrest either (data not shown). Possible explanation for these observations may include that the overexpression level is not high enough to compensate the depletion due to the high molecular weight of CENPE (312 kDa), or the phenotype induced by CENPE deficiency is severe and irreversible. In addition, we cannot exclude the possibility that the effect of XAB2 depletion is mediated by defects in multiple genes as revealed by microarray analysis that a subset of genes involving in cell cycle and mitotic progression are down-regulated.46 for example, CENPE inhibitor like GSK923295,47 syntelin48 or PF272149 is now considered to have antitumour activity. Therefore, it will be interesting to investigate whether XAB2 can serve as a new anti-mitotic target for cancer therapy.Mitosis is one of the critical processes in cell cycle for proper chromosome segregation during cell division. Mitosis dysregulation often causes genome instability or aneuploidy, leads to mitotic catastrophe and cell death, and is closely associated with cancers and many other diseases. Thus, targeting mitosis has been proposed as an attractive therapeutic strategy for cancer therapy,XAB2 construct was purchased from Origene and re-cloned into modified pcDNA3.1 vector  containing HA tag at the 5′ end. A 1355 bp fragment of 5′ region sequence extending from −1263 to 92 (+1 is the transcription start site) of human CENPE gene was amplified by PCR from Hela genomic DNA and cloned into pGL3-Basic vector (Promega) at KpnI/HindIII sites. Deletion constructs of CENPE promoter were amplified from the full length promoter construct using nested PCR. The sequences of all the constructs were confirmed by direct sequencing. Primer sequences are listed in γ-H2A.X) , monoclonal antibodies against HA tag , CyclinB1 , CENPE , and α-tubulin  were used in western blots.Polyclonal antibody against XAB2 , Phospho-Histone H3 (Ser10) , Cdc2 , Histone H2A.X , Phospho-Histone H2A.X . All the cells were cultured in dulbecco's modified eagle medium supplemented with 10% fetal bovine serum.μl siRNA at 50 μM were transfected using Lipofectamine 3000. Cells were treated with CENPE siRNA or XAB2 siRNA for analysis 24 or 55 h, respectively, after transfection. CENPE siRNA and XAB2 siRNA were previously reported,50 non-target siRNA  was used as negative control. The siRNA sequences are as follows: CENPE siRNA (5′-CCACUAGAGUUGAAAGAUAdTdT-3′), XAB2-siRNA-1 (5′-CCAAUUCUCUGUCAAAUGCdTdT-3′), XAB2-siRNA-2 (5′-ACGCAGCACUCUCGAAUUUdTdT-3′).For siRNA mediated gene knockdown, cells were plated in six-well plate and 2 μg of XAB2 shRNA (cloned in PLKO.1), 1.4 μg of pREV, 1.4 μg of pGag/Pol/PRE and 0.7 μg of pVSVG. For control, replace XAB2 shRNA construct with control vector. Six hours after transfection, the mixture was replaced by fresh dulbecco's modified eagle medium containing 10% FBS and cells were cultured for an additional 48 h. Then the medium containing lentiviruses was filtered and stored at −80 °C. As for lentivirus infection, cells grown in six-well plate were infected twice with lentivirus containing 6 μg/ml polybrene for a total of 48 h. For microarray, western blot, immunofluorescence, RT-PCR and cell cycle distribution analysis, cells were harvested after culture in fresh medium for another 24 h. For cell death analysis, cells were harvested 48 h later. The full sequence of XAB2 shRNA-1 and XAB2 shRNA-2 are CCGGGCTTCGCTACATCGAGTTCAACTCGAGTTGAACTCGATGTAGCGAAGCTTTTTG and CCGGGCAGTATGACATGTTCAACATCTCGAGATGTTGAACATGTCATACTGCTTTTTG, respectively. XAB2 shRNA-1 was used in the XAB2 knockdown experiments in this study unless otherwise indicated.For shRNA mediated gene knockdown, lentiviruses expressing XAB2 shRNA were produced as following: 293T cells were placed in 6 cm dish and cotransfected with 1.4 P<0.05 were included in the final results. Functional analysis of the genes with expression significantly changed by XAB2 knockdown was performed using Gene Ontology analysis.Hela cells were plated in six-well plate and infected twice with control or XAB2 shRNA lentivirus. Then cells were harvested and total RNA was isolated using RNeasy plus kit  following the manufacturer's instruction. The integrity of total RNA was monitored with Agilent 2100 Bioanalyzer. Microarrays were performed using Human Transcriptome Array 2.0  with three independent experiments. Results were analysed by using the Transcriptome Analysis Console from Affymetrix. Only genes that had a 2-fold increase or decrease in expression with a significance of To examine the cell cycle distribution, cells were harvested and fixed in 75% ethanol at 4 °C overnight, washed twice with PBS, and then incubated with PI (Sigma) solution containing RNase for 30 min. The flow cytometry analysis was conducted on ACCURIC6, and the data were analysed using FlowJo7.6 software.For cell death analysis, cells were stained using annexin/PI staining kit  for detection according to the manufacturer's instructions.To detect the fraction of phospho-Histone H3 Ser10 positive cells, cells were fixed by 75% ethanol, permeabilized by 0.25% Triton X-100, and then stained with Alexa Fluor488-conjugated phospho-Histone H3 Ser10 antibody  and PI.α-tubulin antibody (1:250) diluted in PBS with 10% calf serum at 4 °C overnight, and immunostained with Alexa 488-labeled anti-mouse antibody (1:500) diluted in the same buffer at room temperature for 30 min, followed by DAPI staining and four washes with PBS for 5 min each. Fluorescence was detected using DMI6000 microscope .Cells were grown in 35 mm cell culture dish with glass bottom , fixed with 4% paraformaldehyde  for 30 min, followed by permeabilization with 0.5% Triton X-100 for 10 min at room temperature. Cells were then incubated with 2.GFP-H2B HeLa cells were a gift from Dr Huiyan Li in China National Center of Biomedical Analysis and were placed in 35 mm cell culture dish with glass bottom (NEST). Twenty-four hours after second infection with control or XAB2 shRNA lentivirus, images were captured every 10 min for 48 h using DMI6000 microscope (Leica) equipped with a × 40 objective lens in a chamber maintained at 37 °C with 5% COCells were seeded into 12-well plate the day before transfection. 1 μg of firefly luciferase constructs were transfected using lipofectamine 2000. The pRL-TK plasmid (100 ng/sample) encoding Renilla luciferase was cotransfected, and the readout was used for normalization of firefly luciferase activity. Cells were harvested 24 h after transfection and luciferase activity was measured with Dual-Luciferase Reporter Assay Kit (Promega). All transfections were performed in triplicate.To assess the effect of XAB2 overexpression or knockdown on CENPE promoter activity, Hela cells in 12-well plates were transfected with XAB2 construct for 24 h or infected twice with lentivirus containing XAB2 shRNA, followed by co-transfection of CENPE promoter construct and pRL-TK vector, cells were then harvested after 24 h for the measurement of luciferase activity.The ChIP assay was performed using EpiQuik Chromatin Immunoprecipitation Kit from Epigentek Group Inc.  according to the manufacturer's protocol. Protein-DNA complexes were immunoprecipitated with HA antibody, and normal mouse IgG was used as negative control. Primer sets for CENPE were shown in t-test with statistical significance defined as *P<0.05, **P<0.01 and ***P<0.001.Data were presented as mean±s.d. (standard deviation). Statistical analyses between two groups were performed using Student's