Saccharomyces cerevisiae), which initiate HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps is unknown. Here we show that yku70Δ cells, which accumulate single-stranded DNA (ssDNA) at the DSB ends independently of Cdk1 activity, are able to repair a DSB by single-strand annealing (SSA) in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by single-strand annealing and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes.Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in haploid cells is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on cyclin-dependent protein kinases  provides an important mechanism to eliminate deleterious lesions, such as DNA double-strand breaks (DSBs). DSB repair by HR uses homologous DNA sequences as a template to form recombinants that are either crossover or noncrossover with regard to flanking parental sequences. Furthermore, a DSB flanked by direct DNA repeats can be repaired by another HR pathway called single-strand annealing (SSA). HR is generally confined to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as repair template. This cell cycle specificity depends on the activity of cyclin-dependent kinases (Cdks), which regulate initiation of HR by promoting nucleolytic degradation (resection) of the DSB ends. Whether Cdks regulate other HR steps is unknown. Here, we show that  DNA double-strand breaks (DSBs) occur spontaneously during DNA replication and after exposure to certain genotoxic chemicals or ionizing radiation. Efficient repair of DSBs can be accomplished by nonhomologous end joining (NHEJ), which directly rejoins broken DNA ends, or by homologous recombination (HR), which utilizes a homologous DNA template to restore the genetic information lost at the break site (reviewed in HR is initiated by 5′-3′ nucleolytic degradation of the DSB ends to yield 3′-ended single-stranded DNA (ssDNA) tails. Replication protein A (RPA) binds to the ssDNA tails to remove their secondary DNA structures, but is then replaced by Rad51 aided by Rad52. Once formed, the Rad51 nucleofilaments search for homologous sequences and then promote invasion of the ssDNA into homologous donor double-stranded DNA to form a joint molecule with a displaced strand (D-loop)  model When a DSB is flanked by direct repeats, its repair primarily occurs by single-strand annealing (SSA). Here, the resected DSB ends anneal with each other instead of invading a homologous DNA sequence , which promote resection of the 5′ DSB ends to yield 3′-ended ssDNA tails that are necessary to initiate HR In RAD9 deletion allows DSB resection in G2 cells that display low Cdk1 activity due the overexpression of the Cdk1 inhibitor Sic1 Indeed, DSB end resection is also negatively regulated by the Yku heterodimer Whether Cdk1 promotes other HR events is unknown. Some evidence suggests that HR steps other than DSB resection might be regulated by Cdk1 activity. For example, formation of Rad52 foci after ionizing radiation (IR) is less efficient in G1 than in G2, suggesting that Cdk1 might control Rad52 recruitment to DSBs Here, we investigate the role of Cdk1 in homology-dependent repair of a DSB. We show that generation of 3′-ended ssDNA at the DSB ends bypasses Cdk1 requirement for the repair of a DSB by either SSA or noncrossover recombination, indicating that Cdk1 is dispensable for these repair events if DSB resection occurs. By contrast, resection is not sufficient to bypass Cdk1 requirement for generating crossover products. Thus, Cdk1 promotes SSA- and noncrossover-mediated recombination by regulating essentially the resection step, while Cdk1 controls further HR steps in order to allow crossover outcomes.YKU70 deletion allows ssDNA generation at DSB ends in G1 cells yku70Δ cells are capable to carry out HR in G1.HR is inhibited in G1 when Cdk1 activity is low, whereas it occurs during S and G2/M cell cycle phases when Cdk1 activity is high YKU70 in a strain where tandem repeats of the LEU2 gene are 0.7 kb apart and one of them (leu2::cs) is adjacent to a recognition site for the HO endonuclease  and remained undetectable in both cultures throughout the experiment  yku70Δ rad9Δ cells. In fact, the 8 kb SSA repair product accumulated in yku70Δ rad9Δ GAL-SIC1ntΔ cells . Upon galactose addition, the HO-induced DSB can be repaired by Rad51-dependent HR using the uncleavable MATa-inc sequence as a donor. This repair event can occur either with or without an accompanying crossover   . These ryku70Δ rad9Δ strains carrying the system in CLB2 under the control of the GAL promoter (GAL-CLB2dbΔ). This Clb2 variant forms active Clb2-Cdk1 complexes also during G1, because it lacks the destruction box, and therefore it is not subjected to B-type cyclin-specific proteolysis Δ overproduction were induced in G1-arrested cell cultures by galactose addition  Δ overproduction were induced in G2-arrested cell cultures by galactose addition, crossover products accumulated, as expected, in yku70Δ rad9Δ cells, but they were undetectable in yku70Δ rad9Δ GAL-SIC1ntΔ cells  except for the presence of a LEU2 fragment inserted, respectively, 0.7 kb or 4.6 kb centromere-distal to leu2::csBAR1 gene, which encodes a protease that degrades the α-factor. Deletions of the YKU70, RAD9, RAD51, RAD52 and BAR1 genes were generated by one-step PCR disruption method. YMV86, YMV45 and tGI354 derivatives strains carrying a fully functional CDC28-HA allele at the CDC28 chromosomal locus were generated by one-step PCR tagging method. A plasmid carrying the GAL-CLB2dbΔ allele was kindly provided by R. Visintin  and was used to integrate the GAL-CLB2dbΔ fusion at the URA3 locus in the tGI354 derivative strains. Strain YLL3019, carrying the GAL-SIC1ntΔ allele integrated at the URA3 locus, was obtained by transforming strain tGI354 rad9Δ yku70Δ with ApaI-digested plasmid pLD1, kindly provided by J. Diffley . The GAL-SIC1ntΔ fusion was cloned into a TRP1-based integrative plasmid that was used to integrate the fusion at the TRP1 locus in the YMV45 derivative strains. Integration accuracy was verified by Southern blot analysis. Cells were grown in YEP medium  supplemented with 2% raffinose (YEP+raf) or 2% raffinose and 3% galactose .Strain genotypes are listed in For Cdk1 kinase assays, protein extracts were prepared as described previously Asp718-SalI fragment containing part of the LEU2 gene as a probe. DSB end resection at the MAT locus in JKM139 derivative strains was analyzed on alkaline agarose gels as described in MATα locus (coordinates 200870 to 201587 on chromosome III). Quantitative analysis of DSB resection was performed by calculating the ratio of band intensities for ssDNA and total amount of DSB products. DSB repair in tGI354 strain was detected as described in MATa band at time zero before HO induction (100%). The repair efficiency (NCO+CO) was normalized with respect to the efficiency of DSB formation by subtracting the value calculated 2 hours after HO induction (maximum efficiency of DSB formation) from the values calculated at the subsequent time points after galactose addition.DSB formation and repair in YMV86 and YMV45 strains were detected by Southern blot analysis using an Table S1Yeast strains used in this study.(DOC)Click here for additional data file.


Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts.A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Castimulus . Here wes (TPCs) , as bein 2+ stores via NAADP/two-pore channels (TPCs) ► Exocytotic granules are themselves acidic Ca2+ stores decorated with TPCs ► TPCs move to the immunological synapse upon TCR activation ► NAADP stimulates exocytosis more efficiently than does IP3/Ca2+ influx alone► The T cell receptor mobilizes acidic Ca All three major Ca2+-mobilizing second messengers—inositol 1,4,5-trisphosphate (IP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP)—increase in concentration following T cell receptor activation [2+ signaling via a recently identified molecular target, the two-pore channel (TPC) [The compartmentation of cell signaling pathways is important for maintaining the fidelity between extracellular stimuli and appropriate downstream responses. For Carocesses . In T ce kinases . We invetivation . Howevertivation , and theel (TPC) , is an o2+ stores in a primary human cytotoxic T lymphocyte (CTL) clone and found pH-neutral as well as acidic Ca2+ stores. Neutral stores were revealed by ionomycin, a Ca2+ ionophore that can only act at neutral organelles [2+-ATPase inhibitor [2+ from acidic organelles also produced Ca2+ responses, i.e., bafilomycin A1 (a V-type H+-ATPase inhibitor), nigericin and monensin (protonophores), and glycyl-phenylalanine 2-naphthylamide  Ca2+ stores [3 and cADPR that target the neutral endoplasmic reticulum (ER).First, using a pharmacological approach, we characterized the intracellular Caganelles , and cycnhibitor    accepte+ stores , thereby2+ signals in CTLs, we indirectly introduced NAADP into the cytosol by bath application of a membrane-permeant ester precursor (NAADP/AM) that is converted to NAADP by intracellular esterases. NAADP/AM elicited Ca2+ oscillations, with the initial spike commonly comprising distinct first and second phases (trans-Ned-19 (2+ release displayed a “bell-shaped” concentration-response curve (2+-free medium (to eliminate Ca2+ influx), NAADP/AM still caused Ca2+ oscillations, thus confirming intracellular stores as the primary Ca2+ source  or ryanodine receptor (RyR), so-called Ca2+-induced Ca2+ release (CICR). We tested for the coinvolvement of the ER, first by depleting the ER with the Ca2+-ATPase inhibitor CPA, which did indeed abrogate NAADP/AM responses , IP3Rs were demonstrably active (3R blockade with 2-APB (2-aminoxydiphenylborate)  A and S2Bllations H. Conver release J–1O, andn the ER .2+ increase: an immediate spike (Ca2+ release from intracellular stores), followed by a slower, sustained increase or Ca2+ oscillations  and high (50 μM) concentrations, respectively (2+ release that interleaves with Ca2+-induced Ca2+ release (IP3Rs) and Ca2+ influx.Having defined NAADP signaling in CTLs, we determined whether it was involved during physiological T cell receptor (TCR) activation. CTLs were stimulated upon engagement with peptide-loaded antigen-presenting cells (APCs), which resulted in the expected two phases of Caectively C. Togeth2+-dependent vectorial exocytosis of cytolytic proteins granzyme B and perforin from CTL cytolytic granules  after APC engagement. Testing for NAADP and acidic Ca2+ stores, we found that Ned-19 or bafilomycin A1 substantially inhibited granzyme B exocytosis , excluding a role for RyRs in exocytosis. In summary, these data implicate NAADP in TCR-stimulated exocytosis.We next asked whether NAADP/acidic stores were important for exocytosis and target cell killing. CTL-dependent killing of target cells can be effected by two processes: (1) the Calysosome ), and . This s kinases . Therefo157–165-HLA A2-restricted TCR 1G4 [2+ responses in Jurkat 1G4 cells that were sensitive to Ned-19 and bafilomycin A1  knockdown —a partial effect consistent with incomplete TPC knockdown  the NAADP/TPC axis was associated with. To that end, we tagged TPC1 and TPC2 with fluorescent proteins and compared their distribution in Jurkat 1G4 cells with other acidic vesicle markers. Both TPC1 A–S4C and2+ domains that selectively couple to exocytosis. That secretory vesicles act as Ca2+ stores has some precedents [That TPCs are found on cytolytic granules agrees with the NAADP acidic store pharmacology E and 1F ecedents .2+ domains required for lytic granule exocytosis [An important characteristic of cytolytic granules is that they rapidly polarize along microtubules toward the immunological synapse (IS) when the TCR is activated J and S4Kocytosis . Moreove2+ for their own exocytosis and deliver Ca2+ in an “autocrine” fashion via TPCs, presumably acting in local perigranular Ca2+ nanodomains [2+ signaling pathways, e.g., IP3, fail to mimic NAADP. Although the Orai1-STIM1 complex (CRAC channels) makes a substantial contribution to CTL Ca2+ signals [2+ influx per se is insufficient to drive exocytosis, requiring the additional presence of protein kinase activators [In summary, TCR activation recruits NAADP to activate target TPC channels resident on the exocytotic granules themselves, and TPCs consequently translocate toward the immunological synapse. Therefore, these granules store and release the Caodomains . In the tivators ; the NAAtivators , but we


The events surrounding Ca2+ mobilization in lymphocytes are tightly regulated and involve the coordination of diverse ion channels, membrane receptors, and signaling molecules. A mechanism termed store-operated Ca2+ entry (SOCE), causes depletion of endoplasmic reticulum (ER) Ca2+ stores following T cell receptor (TCR) engagement and triggers a sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane. The ER Ca2+ sensing molecule, stromal interaction molecule 1 (STIM1), and a pore-forming plasma membrane protein, ORAI1, have been identified as important mediators of SOCE. Here, we review the role of several additional families of Ca2+ channels expressed on the plasma membrane of T cells that likely contribute to Ca2+ influx following TCR engagement, particularly highlighting an important role for voltage-dependent Ca2+ channels (CaV) in T lymphocyte biology.Calcium (Ca In the event of an infection, T lymphocytes, through their TCR, recognize the infectious antigen and are activated and subsequently induced to proliferate and differentiate into effector cells capable of clearing the pathogen. Key components of the signaling events mediating T lymphocyte development, differentiation, homeostasis, effector function, and cell death are the universal second messenger calcium  and the associated hydrolysis of phosphatidylinositol-3,4-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) causing release of ER Ca2+ stores into the cytoplasm. During the event of store-operated Ca2+ entry (SOCE), depletion of ER Ca2+ stores triggers a sustained influx of extracellular Ca2+ through Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane  by an antigen processing cell. In primary immune responses, this is the function of dendritic cell (DC). DCs take up soluble and particulate antigen as well as cellular debris by phagocytosis, endocytosis, or macropinocytosis and degrade them in endolysosomal compartments where liberated foreign antigens, usually peptides, are subsequently loaded onto MHC-I or MHC-II molecules that cycle to the plasma membrane. Here, the MHC/foreign antigen complex is recognized by a cognate TCR expressed on a specific T lymphocyte , Ca2+-calmodulin-dependent kinase (CaMK) and its target cyclic AMP-responsive element-binding protein (CREB), myocyte enhancer factor 2 (MEF2) targeted by both calcineurin and CaMK, and nuclear factor kappa B (NFκB)  pathway in T cells. RasGRP that activates Ras not only has a DAG binding domain but also has a pair of EF-hand motifs that can directly bind Ca2+  , similar to the ER-associated Ca2+ channels, have been shown to be expressed at the plasma membrane of T cells. In addition, T cell expressed adenosine triphosphate (ATP) responsive purinergic P2 (P2X) receptors and glutamate mediated N-methyl-d-aspartate (NMDA) activated receptors have shown significant Ca2+ permeability. Finally, voltage-dependent Ca2+ channels (CaV), the focus of this review, have been identified to play a crucial function in T cells . When ER Ca2+ stores are depleted, the EF-SAM domain interaction in STIM1 becomes unstable resulting in the oligomerization of STIM1 molecules , invariant natural killer T cells and TCRαβ+ CD8αα+ intestinal intraepithelial lymphocytes, owing to a defect in IL-2 or IL-15 signaling has been noted in STIM1- and STIM2-deficient mice  and hepatosplenomegaly (enlarged liver and spleen) as well as autoimmune hemolytic anemia and thrombocytopenia resulting from immune responses directed against the red blood cells and platelets, respectively  channels ; the melastatin TRPs (TRPMs); the mucolipins (TRPMLs); the polycystins (TRPPs); and ankyrin transmembrane protein 1 (TRPA1) , nicotinamide adenine dinucleotide (NAD+), hydrogen peroxide (H2O2), and cyclic ADPR  and low-voltage activated (LVA) channels. Further analysis of the CaV channels allowed for additional classification of the channels into groups with distinct biophysical and pharmacological properties: T (tiny/transient)-, N -, P/Q (Purkinje)-, R (toxin-resistant)-, L (long-lasting)-type channels  each composed of six transmembrane segments (S1–S6) with a re-entrant pore-forming loop (P-loop) between S5 and S6. The P-loop contains four highly conserved negatively charged amino acids responsible for selecting and conducting Ca2+ while the S6 segments form the inner pore , a wide drug class that can either activate (for example: Bay K 8644) or inhibit (for example: nifedipine) the activity of the channel  distinct from the retina  may result in CaV1.4-mediated Ca2+ influx from outside the cell, allowing the filling of intracellular stores and the initiation of a pro-survival program. This recent data supports the concept that in the absence of CaV1.4, there is a reduction in the influx of extracellular Ca2+ coupled to self/MHC-TCR interaction, resulting in low cytoplasmic Ca2+ levels and depleted Ca2+ ER stores  ryanodine receptors (RyRs) in excitable cells  and CaV1.2 and ORAI1 has been described  to interact with the C-terminus of ORAI1 and CaV1.2 channels. ORAI1 channels are activated by STIM1 and subsequently open causing sustained Ca2+ influx from the extracellular space. Conversely, STIM1 inhibits Ca2+ influx through CaV1.2 and promotes its internalization, further shutting down the activity of the channel (Park et al., V1 channels (Figure V and co-ordinately inhibiting ORAI1. This results in CaV1-mediated Ca2+ influx from outside the cell, filling of depleted intracellular stores, and induction of a signaling cascade to activate a pro-survival program within the naïve T cell. The activation and inhibition of CaV1 channels through STIM1 or other TCR-mediated events is an intriguing concept and will likely be the focus of many new studies.Recently, an interesting reciprocal relationship between CaV1 function is vital for T cell Ca2+ signaling, their specific functions have yet to be fully explored. Further work is required to clarify the role played by each CaV1 channel family member as well as the other Ca2+ channels in shaping the Ca2+ signal. Altogether, these studies do provide a new framework for understanding the regulation of lymphocyte biology through the function of several Ca2+ channels, particularly the L-type Ca2+ channels, in the storage of intracellular Ca2+ and operative Ca2+ regulation during antigen receptor-mediated signal transduction.Although Ca2+ channel biology have direct implications in designing new modalities for modifying T cell responses using drugs that are known to control Ca2+ channels activities, such as the plethora of drugs that already exist for modifying CaV1 channels. Agents that target the CaV1 splice variants expressed in lymphocytes and inhibit the activity of the channel may serve as more specific immunosuppressants than the current options. Relevant applications for these agents may include therapy for autoimmune diseases, reduction of transplant rejection risk, and treatment of other disorders requiring suppression or in the case of existing immunodeficiency, activation of the immune system.Overall, the translational aspects of the current research in the field of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We therefore evaluated its potential as a cardioplegia additive.The Na2+ levels were measured using Ca2+ sensitive dye (fura-2 AM). Isolated rat hearts were arrested using cardioplegia with (n = 7) or without (n = 6) SEA0400 (1 μM) then reperfused after 45 min of ischemia. Left ventricular (LV) function, troponin release, and mitochondrial morphology were evaluated.Isolated rat cardiomyocytes were exposed to hypoxia (45 min) followed by reperfusion. During hypoxia, cells were protected using cardioplegia with (n = 25) or without (n = 24) SEA0400 (1 μM), or were not protected with cardioplegia . Intracellular Ca2+ (305 ± 123 nM after 20 minutes of ischemia). Increases in intracellular Ca2+ were reduced in cells arrested with cardioplegia without SEA0400; however cardioplegia containing SEA0400 was associated with the lowest intracellular Ca2+ levels . Hearts arrested with cardioplegia containing SEA0400 had better recovery of LV work compared to cardioplegia without SEA0400 . Troponin release during reperfusion was lower , and there were more intact , and fewer disrupted mitochondria  in the SEA0400 group.Cardiomyocytes exposed to hypoxia without cardioplegia had poor survival (13%). Survival was significantly improved when cells were protected with cardioplegia containing SEA0400 ; cardioplegia without SEA0400 was associated with intermediate survival (42%). Cardiomyocytes exposed to hypoxia alone had a rapid increase in intracellular Ca2+ during ischemic arrest in isolated cardiomyocytes and prevents myocardial injury and improves recovery of LV function in isolated hearts.SEA0400 added to cardioplegia limits accumulation of intracellular Ca Perfusion was interrupted and cardioplegia delivered through the aorta (20 mL/kg body weight). Cardioplegia was prepared by mixing base solution used in clinical practice  with the shed blood in a ratio of 4 parts blood to 1 part base solution resulting in a final cardioplegia solution with an approximate ionic composition of (mM): 136 Na+, 19 K+, 5 Mg2+, and 1 Ca2+. SEA0400  or its vehicle  was added to the cardioplegia, which was cooled on an ice bath (≈4°C) and bubbled with oxygen prior to delivery. After cardioplegia delivery, the heart was exposed to ambient room temperature (22 ± 0.2°C) for the duration of the ischemic period (45 min). After reperfusion with 37°C oxygenated Kreb’s solution (20 min), LV function data were again collected.Rats were assigned to their experimental group before the experiment started. Hearts were attached by the aorta to a Langendorff apparatus . Shed blood was collected for mixing with the cardioplegia solution. Electrocardiogram and aortic pressure were recorded continually. Coronary flow was measured using a transit time ultrasound probe  on the inflow cannula, and coronary vascular resistance was calculated by dividing aortic pressure by flow. A custom non-compliant balloon tipped catheter was placed through the mitral valve into the LV cavity. Hearts were perfused with oxygenated Kreb’s solution  during reperfusion were quantified.Myocardial damage was assessed by measuring troponin I release from timed collections of coronary sinus effluent during reperfusion . Mitochondria were isolated from LV muscle and a blinded observer categorized and counted intact, swollen, and disrupted mitochondria from random electron photomicrographs from each heart ,24.Data are presented as mean ± SEM. Tests for statistical significance included Kaplan-Meier log-rank survival analysis, unpaired t-test, and one-way and two-way repeated measures ANOVA with post-hoc comparisons by Newman-Keuls and Holmes-Sidak method respectively.We examined the impact of simulated cardioplegia containing the NCX blocker SEA0400 on cell survival during ischemia and reperfusion in isolated cardiomyoyctes Figure . Cell su2+ levels were measured in isolated cardiomyocytes during ischemia and reperfusion. Ischemia without cardioplegic protection was associated with a rapid increase in both diastolic and systolic intracellular Ca2+ levels  by 20 minutes into the ischemic period (p < 0.001). Intracellular Ca2+ accumulation was limited in cells arrested with cardioplegia . Diastolic Ca2+ levels rose least in cells protected with cardioplegia containing SEA0400 from 62 ± 5 nM at baseline to 110 ± 17 nM (77%) at the end of the ischemic period . While diastolic Ca2+ levels returned towards baseline values soon after reperfusion in both groups protected with cardioplegia  in hearts arrested with cardioplegia not containing SEA0400 Figure A. HoweveWe quantified reperfusion arrhythmias (ventricular tachycardia and fibrillation) after cardioplegic arrest. All hearts had one or more episodes of ventricular arrhythmia. All episodes terminated spontaneously. The onset of reperfusion arrhythmias was delayed in the SEA0400 group  but the resolution of arrhythmias occurred at a similar time in each group . The number of distinct episodes of arrhythmia was 16 ± 3 episodes in the group not treated with SEA0400 and 7 ± 3 episodes in the SEA0400 group, a difference that may have been due to chance (p = 0.054).-1) in hearts arrested with cardioplegia without SEA0400 but was only 109 ± 9% of baseline (7.0 mmHg.min.ml-1) in hearts protected with cardioplegia containing SEA0400 (p = 0.0088). The proportion of intact mitochondria was lower and the proportion of disrupted mitochondria was higher in isolates from LV myocardium after cardioplegic arrest and reperfusion when compared to normal myocardium not subjected to ischemia .In isolated cardiomyocytes, we found that simulated cardioplegia limited the development of intracellular Cae degree ,25 which2+ influx) at or below this concentration [2+ influx during ischemia, potential blockade of forward mode NCX or unwanted effects on other ion channels (L-type Ca2+) during reperfusion may be problematic.We examined only one dose level of SEA0400 (1 uM), which was chosen based on previous studies that demonstrated relatively specific targeting of the reverse mode NCX activity . While ischemia-reperfusion injury is primarily initiated by Caerfusion . Future 2+ overload during cardioplegic arrest. This is associated with reduced myocardial and mitochondrial damage and improved recovery of LV function in the whole heart. SEA0400 does not appear to increase reperfusion arrhythmias when used as a cardioplegia additive. Blockade of NCX activity with SEA0400 in cardioplegia is a promising strategy to improve myocardial protection during cardiac surgery and warrants further study to confirm the mechanism of action and to evaluate its benefit in human cells and a whole animal preclinical model.In isolated cardiomyocytes, SEA0400 limits Ca+/Ca2+ exchanger; ELISA: Enzyme-linked immunosorbent assay; LVDP: Left ventricular developed pressure.LV: Left ventricle; NCX: NaSEA0400 used in this study was donated by Taisho Pharnaceutical Co, Ltd., Tokyo, Japan. The authors had full control of the design of the study, methods used, outcome parameters and results, analysis of data, and production of the written report. The authors have no financial interests to disclose.JE carried out isolated cardiomyocyte studies. These were performed in the lab of SH. AA carried out the isolated heart studies. All authors were involved in the design of the studies. SO coordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Imaging studies demonstrate a Na+ influx that then causes depolarization-mediated activation of voltage-gated Ca2+ channels (CaV), which allows Ca2+ influx upon divalent re-introduction. Inhibition of CaV  or NaV  fails to reduce the Na+ influx. The Ca2+ influx is inhibited by CaV inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg2+ or Ca2+ alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg2+ and Ca2+ from external solutions evokes a large slowly-inactivating voltage-gated current (DFI) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca2+ alone fails to evoke DFI. Evidence suggests DFI is a non-selective cation current. The DFI is not reduced by inhibition of NaV , CaV , KV  or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca2+ (IC50∼0.5 µM) or Mg2+ (IC50∼3 µM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg Sensory vagal afferent nerves express specific ion channels that are gated by mechanical, thermal, osmotic, acid/base and chemical stimuli; activation of these ion channels leads to membrane depolarization and the initiation and propagation of action potentials centrally towards the CNS 2+, Mg2+) interaction is known to modulate the function of both soluble and structural proteins, including enzymes, membrane receptors, ion channels and transporters. Extracellular divalent cations have been previously shown to modulate excitability in neurons and other excitable cells. Extracellular divalent cations contribute substantially to the surface potential of the plasma membrane, effectively modulating the gating of voltage-gated ion channels . Increasing divalent concentrations increases this charge screening effect, which results in slower gating kinetics and rightward shifts in voltage-dependent activation curves VV2+ blocks acid-sensing ion channels 2+ blocks NMDA channels 2+ channel + channels − channels Divalent cation  on K+ and Cl− channels 2+ with Mg2+V or CaV. The finding that reductions in external Ca2+ activates vagal afferents is surprising as it is fairly common practice in Ca2+ imaging studies of dissociated neurons to use Ca2+-free external solutions (chelated with EGTA) to determine the extent to which Ca2+ influx or Ca2+ intracellular stores contribute to an increase in [Ca2+]i during neuronal activation. In these imaging studies EGTA (Ca2+ depletion) does not produce any overt neuronal activation, which is contradictory to the Undem et al study 2+ chelator. In the present study, we sought to clarify the Ca2+/divalent sensing of vagal sensory neurons and evaluated the effect of Ca2+ and Mg2+ on dissociated mouse vagal neurons using live cell imaging and electrophysiology. The data indicate the presence of a high affinity divalent sensor on vagal neurons that is coupled to a novel voltage-gated non-selective cation current.Extracellular divalent concentrations are not fixed , and are subject to change due to disease and diet. The effect of divalent concentration variations on sensory nerve excitability is unclear. Studies suggest that extracellular CaAll experiments were approved by the University of South Florida Institutional Animal Care and Use Committee. In total 120 mice and 2 guinea pigs were used in this study.Mouse sensory ganglia  were isolated and enzymatically dissociated from wild-type C57BL/6J mice using previously described methods 2) in DMEM (containing 110 mg/l pyruvate and 564 mg/l L-Glutamine) supplemented with 10% FBS and 0.5% penicillin/streptomycin. Cells were removed from their culture flasks by treatment with Accutase (Sigma), then plated onto poly-D-lysine-coated cover slips and incubated at 37°C for >1 h before experimentation.Wild-type HEK293 cells were obtained from the sources described by Taylor-Clark et al Fluorescent imaging: Cells were studied for changes in [Ca2+]i with Fura 2AM  or [Na+]i with SBFI . Neuron-covered coverslips were incubated (at 37°C) with Fura-2 AM  or SBFI  in L-15 media containing 10% FBS. HEK293-covered coverslips were incubated (at 37°C) with SBFI  in DMEM (containing 110 mg/L pyruvate and 564 mg/L L-Glutamine) supplemented with 10% FBS. For imaging, the coverslip was placed in a custom-built heated chamber (bath volume of 300 µL) and superfused by gravity at 6 ml/min with Locke solution : 136 NaCl, 5.6 KCl, 1.2 MgCl2, 2.2 CaCl2, 1.2 NaH2PO4, 14.3 NaHCO3, 10 dextrose ) for 15 minutes before and throughout each experiment. Changes in [Ca2+]i (Fura 2AM) or [Na+]i (SBFI) were monitored by sequential dual excitation, 340 and 380 nm (emission 510 nm), measured by digital microscopy  and analyzed by specialized software . The ratio images were acquired every 6 or 12 seconds. At the end of the dissociated neuronal studies, neurons were exposed to KCl  to confirm voltage sensitivity. At the end of all Fura 2AM experiments, cells were exposed to ionomycin  to obtain a maximal response. At the end of all SBFI experiments, cells were exposed to gramicidin  to obtain a maximal response. In those studies in which external Na+ was completely replaced by N-methyl-D-glucamine (NMDG+), the cells were superfused with (in mM) 154 NMDGCl, 4.7 KCl, 1.2 MgCl2, 2.2 CaCl2, 10 HEPES, 5.6 dextrose (pH (7.4) with 1.5 M NMDG). External NMDG+ was then replaced with equimolar Na+ prior to gramicidin positive control.2+]i or [Na+]i, we used the excitation ratio 340/380  and relate all measurements to the peak positive response in each viable cell. Only cells that had a robust response to the positive control were included in analyses. At each time point for each cell, data was presented as the percentage change in 340/380 ratio (R), normalized to maximum response : responsex = 100×(Rx−Rbl)/(Rmax−Rbl), where Rx was the 340/380 ratio of the cell at a given time point, Rbl was the cell's mean baseline 340/380 ratio measured over 120 s, and Rmax was the cell's peak 340/380 ratio. All data is presented as the normalized mean ± S.E.M of all the neurons: as a % of ionomycin or gramicidin for [Ca2+]i or [Na+]i studies, respectively. External divalent cations were chelated by a 3 minute treatment of 5 mM EDTA (with 0 mM Mg2+ and 0 mM Ca2+ added). External Ca2+ was chelated by a 3 minute treatment of 5 mM EGTA (with 1.2 mM Mg2+ and 0 mM Ca2+ added).For the analysis of free), 35 TEA and 1 4-AP, adjusted to pH 7.2 with NaOH. Cells were superfused with HEPES-buffered bath solution : 140 NaCl, 4.7 CsCl, 1.2 MgCl2, 2.5 CaCl2, 10 HEPES, 10 dextrose, 5 TEA, 1 4-AP adjusted to pH 7.4 with NaOH). For experiments in low Na+, 108 mM mannitol was substituted for 54 mM NaCl in the bath solution.Recordings from HEK293 and vagal neurons were made using whole-cell or perforated patch-clamp techniques as indicated. Perforation was achieved using 5 µg/ml gramicidin. Patch pipettes were fabricated from 1.5 mm o.d. borosilicate glass (Sutter Instrument Co.) and fire-polished. Pipettes for perforated patch recording (1–3 MΩ) were filled with solution composed of (mM): 140 KCl, 1 CaClV-like current (peakI) was measured as the peak current observed in the first 6 ms of the voltage step, and the ‘persistent’ current (persistentI) measured at 24.5 ms from the start of the step. For experiments examining the time-dependent inactivation of DFI, cells were held at −120 mV and stepped to −20 mV for 25, 525, 1025, 1525 and 2025 ms. In order to minimize the effect of NaV contribution to the overall current, a number of experiments were also performed by holding cells at −120 mV, applying a 200 ms long step of −20 mV and stepping in 20 mV increments from −120 to +40 mV. Tail currents were fitted using the following equation: Membrane currents were recorded using a MultiClamp 700B amplifier, Digidata 1440A and pClamp 10 acquisition software (Molecular Devices). Current signals were sampled at 100 KHz and filtered at 5 KHz. Cells were routinely voltage-clamped at −120 mV and a series of depolarizing voltage steps was applied in 10 mV increments from −120 to +60 mV. Currents evoked by voltage steps were analyzed at two different time points: the peak Na2+ was chelated using 5 mM EGTA. In some experiments external nanomolar and micromolar Ca2+ was clamped at the desired concentration using 5 mM EGTA with the added Ca2+ calculated by Maxchelator (http://www.stanford.edu/~cpatton/xlsconstants.htm). All solutions were made with de-ionized water, nominally free of divalent cations. All purchased salts were of the highest purity to reduce to the fullest extent any contamination with divalent cationsExternal divalent cations were chelated using 5 mM EDTA. External CaSKF-96365 and cilnidipine were purchased from Tocris . Fura 2AM and SBFI-AM were purchased from TEFLabs . Tetrodotoxin and ω-conotoxin were purchased from Alomone (Israel). All other chemicals were purchased from Sigma-Aldrich .2+ following 10 mM EDTA treatment of guinea pig trigeminal neurons produced substantial influx of Ca2+, as measured by Fura 2AM 2+ imaging (Fura 2AM). Treatment with 5 mM EDTA  for 3 minutes led to a slow and minor decrease in [Ca2+]i. Upon re-introduction of divalents , an increase in [Ca2+]i was observed in 46 of 52 vagal neurons. The neurons were further characterized by their response to capsaicin  agonist) – a hallmark of nociceptive C-fiber nerves involved in the detection of noxious stimuli 2+, 0 mM Mg2+) were observed in trigeminal neurons  and DRG neurons   As part of a previous study, we had noted that the re-introduction of 2.2 mM Ca2+ in the responses of vagal neurons to external divalent cation withdrawal. EDTA chelates all divalent  cations with high affinity, whereas EGTA chelates all divalent  cations except Mg2+ with high affinity 2+ with EGTA and substitute varying [Mg2+]. Upon re-introduction of divalents  following complete divalent chelation with EDTA, vagal neurons responded with a mean increase in [Ca2+]i of 50.4±3.1% of ionomycin (n = 94). In contrast, vagal neurons failed to responded to re-introduction of 2.2 mM Ca2+ following Ca2+ chelation with EGTA  and 0 mM  resulted in a progressive increase in [Ca2+]i following re-introduction of divalents, indicating that withdrawal of Mg2+ is critical to these responses. To test whether or not withdrawal and re-introduction of Mg2+ is sufficient to elicit an increase in [Ca2+]i, we compared responses of vagal neurons to 3 min treatment of 0 mM Mg2+ (trace), with and without chelation of Ca2+ with EGTA. Withdrawal and re-introduction of Mg2+ alone had no significant effect on [Ca2+]i (n = 21), indicating that withdrawal of Ca2+ is also critical to these responses  are Ca2+ permeable plasma membrane ion channels that are typically activated following depletion of Ca2+ stores in the endoplasmic reticulum through the actions of Ca2+ sensor proteins (STIM) 2+] and [Mg2+] 2+]i during re-introduction of divalents  following treatment with EDTA  and 10 µM cilnidipine (N-type and L-type blocker) significantly reduced the response of vagal neurons to the re-introduction of divalents  following treatment with EDTA  from 50.4±3.1% (n = 94) to 16.2±2.2% of ionomycin   through CaV is first dependent on the influx of Na+. It is likely that the increase in [Ca2+]i observed is a consequence of neuronal depolarization (Na+ influx) occurring during the withdrawal of external divalent cations (EDTA treatment).Vagal sensory neurons contain many Ca<0.0001) . Similar n = 45; . Taken t2+. Vagal neurons were treated with Mg2+-free buffer containing various Ca2+ concentrations (titrated by EGTA chelation). Treatment of vagal neurons with sub-millimolar Ca2+ (and zero Mg2+) evoked two distinct responses (2+ concentrations (10 nM and 100 nM), Ca2+ influxes occurred upon re-introduction of divalents, similar to EDTA-induced responses. Increasing the external Ca2+ concentration reduced this ‘addback’ response. However, treatment with low micromolar Ca2+ (and zero Mg2+) evoked an immediate Ca2+ influx that was not significantly increased following the re-introduction of millimolar Ca2+ (suggesting that the CaV are not activated during the re-introduction). Further increasing the Ca2+ concentration reduced this ‘immediate’ response so that treatment with 2.2 mM Ca2+ (and zero Mg2+) failed to evoke either responses  of dissociated mouse vagal sensory neurons, we found that treatment with EDTA  induced an increase in [Na+]i in 92 of 106 neurons: mean response 78.4±2.5% of gramicidin , indicating that Na+ influx occurred as a result of both Ca2+ and Mg2+ depletion  also failed to activate vagal neurons (n = 25), indicating that neuronal activation was specifically dependent on divalent chelation rather through some other action of EDTA itself.Using ratiometric Naamicidin . To conf n = 33; . As withepletion . In addi+ influx following external divalent depletion. Vagal sensory neurons express a number of NaV, including tetrodotoxin (TTX)-sensitive NaV1.7 and the TTX-resistant NaV1.8 and NaV1.9 V currents + influx (n = 39).We next evaluated the contribution of voltage-gated channels in the NaV are able to conduct Na+ ions in the absence of external Ca2+2+ influx following re-introduction of divalents was reduced by CaV inhibition, we tested the role of these channels in the Na+ influx. Pretreatment of the neurons with a combination of 1 µM ω-conotoxin (potent N-type channel toxin) and 10 µM nifedipine no significant effect on EDTA-induced responses .Previous studies including those of chick DRG neurons have demonstrated that Ca n = 45; . In addi2+ and Na+ imaging studies of dissociated mouse neurons indicate that chelation of both Ca2+ and Mg2+ is required for the influx of Na+ which (likely through depolarization) leads to the activation of CaV that then allows for Ca2+ influx upon re-introduction of external Ca2+. Inhibition studies suggest that the Na+ influx in response to external divalent chelation is not via SOC, TRPM7, NaV or CaV channels. In order to evaluate whether or not the EDTA-induced Na+ influx was a non-specific effect on membrane integrity we compared the responses of mouse vagal neurons with HEK293 cells in our SBFI assay. Unlike vagal neurons, HEK293 failed to demonstrate Na+ influx in response to a treatment with EDTA , these pulses evoked a complex current: an NaV-like fast multi-component inward current with fast inactivation and an extrapolated reversal potential (+90 mV) consistent with Na+ selectivity – likely due to the activation of NaV1.7 and 1.8; and a uncharacterized smaller slowly inactivating persistent current with a reversal potential at approximately +30 mV. These currents are consistent with other studies of sensory neurons 2+, 0 mM Mg2+) caused dramatic changes in the current profile until the EDTA was washed away and the divalents were re-introduced, at which point the currents returned to baseline  also evoked a large voltage-gated ‘persistent’ current in all neurons tested . The DFI was apparent either when using gramicidin (5 µg/ml) to obtain perforated patches  or in whole-cell patch-clamp recordings (n = 24). We briefly repeated these studies in guinea pig vagal neurons using whole-cell patch-clamp recording and found a similar voltage-gated persistent DFI . Finally, to determine if the evoked DFI was a non-specific effect on membrane integrity or patch pipette seal resistance, we performed whole-cell patch-clamp electrophysiology on HEK293 cells. 5 mM EDTA treatment failed to evoke a significant voltage-activated DFI current in HEK293 cells , indicating that the DFI observed in sensory neurons is likely due to the selective expression of specific divalent-sensitive molecular identities.To further characterize the response of mouse vagal neurons to chelation of external divalent cations we used whole-cell patch-clamp electrophysiology. In order to eliminate Kbaseline . The NaVthdrawal : peak ac2+ and Mg2+ in the evoked DFI in vagal neurons. As with the SBFI Na+ imaging data, treatment with 5 mM EDTA saturated with divalents  failed to activate the neurons . Consistent with a requirement for both Ca2+ and Mg2+ depletion, EGTA  also failed to evoke a significant DFI (n = 3) – although we nonetheless observed a shift in the NaV-like current profiles consistent with a reduction in charge screening ). Decreasing the [Mg2+] in the Ca2+-free EGTA external solution evoked currents similar to EDTA (2+ was on occasion increased from 30 to 60 s (3/6 cells). Consistent with our Fura 2AM Ca2+ imaging data, where external [Mg2+] greater than 200 µM effectively inhibited Ca2+ influx, 100 µM Mg2+ (and 0 mM Ca2+) produced noticeably smaller DFI currents compared to cells treated with EDTA . There was little difference between the Mg2+ inhibition of DFI at −70 mV and +60 mV , which suggests that Mg2+ external pore block is not the predominant mechanism by which Mg2+ inhibits DFI.We next evaluated the role of Ca to EDTA . It shouDFI were briefly studied. The voltage-dependence of DFI was tested by holding vagal neurons at either −120 mV or 0 mV, then stepping to +60 mV for 25 ms. We studied the evoked outward current in control conditions and with EDTA  treatment. Chelation of external divalents evoked a robust outward current in steps from −120 mV but not in steps from 0 mV (DFI had undergone voltage-dependent inactivation. The rate of inactivation was examined by subjecting cells to a series of depolarizing steps to −20 mV (from −120 mV) of increasing duration (25 ms to 2025 ms) and, following the return to −120 mV, inward tail currents were recorded. Under control conditions tail current amplitude decreased over time to a small plateau consistent with the presence of NaV1.8 2+, 0 mM Mg2+) tail current amplitude from the evoked DFI decreased over time , indicating that the DFI was indeed slowly inactivating .The inactivation characteristics of rom 0 mV . These rtivating . Upon reV-like currents, likely due to the expression of NaV1.7 and NaV1.8. Unlike NaV1.7 that can be effectively blocked by the selective toxin TTX, there are no selective and completely effective blockers of NaV1.8. In order to gain more information on the I–V relationship of DFI we employed a long depolarizing pulse (to −20 mV from −120 mV for 200 ms) prior to a series of voltage steps in order to reduce to the fullest extent (through inactivation) the NaV-like currents. These pulses should allow for the characterization of the current passed by the activated divalent-sensitive channel (with the assumption that a significant portion of the channels have not inactivated after the 200 ms depolarization). Treatment with EDTA  evoked a current that reversed at approximately +13 mV  increase in divalent chelation by EDTA (http://www.stanford.edu/~cpatton/xlsconstants.htm).Mouse vagal ganglia demonstrated complex Nay +13 mV . When exV and NaV and TRPM7 in the DFI was assessed. Previous studies have demonstrated that in the absence of external Ca2+, CaV can conduct monovalent cations (in particular Na+) DFI in control neurons with neurons exposed to combined treatment of 10 µM nifedipine and 10 µM cilnidipine, which substantially reduced depolarization-induced Ca2+ influx in Fura 2AM studies  caused a 65% reduction in the peak NaV-like current evoked by 25 ms depolarizing steps, consistent with the presence of multiple NaV channels ). However, lidocaine failed to inhibit the DFI  has been shown to inhibit numerous NaV currents including ‘persistent’ currents V inactivation, thus we employed a 200 ms pre-pulse step from −120 mV to −20 mV prior to a series of voltages. 100 µM riluzole failed to reduce the DFI  vagal neurons were treated with EDTA  until the DFI was detected, then 20 µM spermine (TRPM7 inhibitor) was added to the external solution. Consistent with the lack of effect of spermine on the EDTA-induced Ca2+ influx  has/have large conductance or (2) expressed in high numbers or both.This study demonstrates that a reduction of external divalent cations to micromolar concentrations activates mammalian sensory nerves via the gating of a voltage-dependent non-selective cation current. At present it is unclear if this current is produced by a single channel population or multiple channel populations, although our data describe a channel(s) that is/are activated by depolarization and undergoes time- and voltage-dependent slow inactivation. Judging from the substantial current density measured (>200 pA/pF) following current activation, we predict that the putative channel(s) underlying the 2+ and Mg2+ depletion for any neuronal activation to occur. We have shown in Ca2+ imaging, Na+ imaging and patch-clamp electrophysiology that ‘complete’ chelation of Ca2+ by EGTA with normal Mg2+ (1.2 mM) fails to evoke the current. Similarly, reduction of Mg2+ to trace concentrations (estimated to be <1 µM) failed to evoke significant responses. When both Ca2+ and Mg2+ were depleted, however, over 90% of neurons responded. Our data show that the divalent sensor can be almost completely inhibited by [Ca2+]o≥100 µM (IC50∼0.5 µM) or by [Mg2+]o≥200 µM (IC50∼3 µM). All our sub-millimolar Ca2+ concentrations were titrated with either EGTA or EDTA so it is theoretically possible that the neuronal activation was due to a depletion of other trace multivalent cations present in the solution (i.e. independent of Ca2+). Both EGTA and EDTA chelate multivalent cations (with the obvious exception of Mg2+ not being chelated by EGTA) with very high affinity (http://www.stanford.edu/~cpatton/xlsconstants.htm). However, our studies of neuronal responses to various concentrations of external Ca2+  have affinities for EGTA several orders of magnitude higher than Ca2+. Therefore the inhibition of neuronal activation must be dependent on Ca2+ specifically.A striking characteristic of this study is the absolute requirement for both Canal Ca2+ , suggest2+ we observed Na+ influx, which was absent when external Na+ was replaced with the large cation NMDG+. In unclamped neurons Na+ influx induced the activation (likely through depolarization) of voltage-gated CaV that are expressed in all sensory neurons. Activated L-type and N-type CaV do not undergo rapid inactivation and so, upon re-introduction of Ca2+ into the external solutions, they were able to conduct a substantial Ca2+ influx (the ‘addback’ response). This response was eliminated by combinations of CaV inhibitors and also by Na+ replacement with NMDG+. Crucially, inhibition of CaV failed to prevent the Na+ influx, confirming that the ‘addback’ Ca2+ influx through CaV was triggered by Na+ influx through a CaV-independent mechanism. Whole-cell patch-clamp studies support the hypothesis that this channel is permeable to Na+ ions as the inward current at negative potentials was reduced when external NaCl was partially replaced with mannitol. With our solutions the reversal potential of the DFI (near zero) suggests that the putative channel(s) is also permeable to cations other than Na+: outward currents were observed using either Cs+ (e.g. + (data not shown) in the pipette solution. In addition our Fura 2AM experiments suggest that the putative channel(s) is Ca2+ permeable (When the putative channel(s) is activated in the absence of external CaCs+ e.g.  and 7 orermeable .+ current in over 95% of vagal sensory neurons. Vagal sensory neurons express voltage-gated NaV 1.6, 1.7, 1.8 and 1.9 + currents evoked by neuronal depolarization (V are not responsible for the DFI: 1) The DFI was not inhibited by high concentrations of lidocaine or riluzole, the Na+ influx (SBFI recordings) was not inhibited by high concentrations of lidocaine or TTX, and the Ca2+ influx upon re-introduction of divalents (Fura 2AM recordings) was not inhibited by TTX (data not shown). 2) The DFI has a near zero reversal potential inconsistent with very high Na+ selectivity. 3) Peak NaV current amplitude was unchanged by a reduction of external divalent cations. 4) The inactivation kinetics of DFI suggest very slow inactivation over seconds rather than milliseconds  We observed charge screening effects on the NaV-like currents but failed to detect DFI when neurons were treated with Ca2+-free solutions containing 1.2 mM Mg2+. 6) The DFI was absent in HEK293 cells transiently transfected with human NaV1.7 (data not shown).Depletion of external divalent cations evoked a substantial voltage-gated Narization . PreviouV-like currents precluded precise analysis of the DFI kinetics, which likely must wait until the putative channel(s) has been cloned. Other known channels were investigated for their role as the divalent sensor in mouse vagal neurons: DFI was not reduced by either a combination of 4AP and TEA (KV inhibitors) nor by a combination of cilnidipine and nifedipine (CaV inhibitors), and the EDTA-induced Na+ influx was not reduced by a combination of ω-conotoxin and nifedipine (CaV inhibitors), suggesting that DFI is not dependent on KV or CaV. Lastly, inhibition of SOC or TRPM7  failed to block EDTA-induced responses (DFI as these channels are only expressed in a subset (approximately 60%) of vagal neurons, and DFI was observed in almost all neurons studied. This is supported by the fact that ruthenium red, which also inhibits TRPV1 and TRPA1 + influx.Nevertheless, the presence of Naesponses , 8D. It 2+ evoked transient non-selective cation currents that were inhibited by verapamil but not by external Mg2+ in chick DRG neurons V that was inhibited by either external Ca2+ or Mg2+2+ with Mg2+ in guinea pig vagal neurons V blockers 2+, Reductions in extracellular divalent cations have produced non-selective cation currents in other studies. A reduction of CaV, KV, CaV, SOC and TRPM7 channels. The physiological relevance of these ion fluxes on vagal neuronal excitability is not known at this time. Extracellular Ca2+ and Mg2+ concentrations are not fixed in vivo  and can be substantially decreased in ischemia + influxes as a result of decreased divalent cation external solutions that are capable of triggering voltage-gated Ca2+ channel activation, indicating physiologically relevant depolarizations of these neurons.In summary, we identified a novel voltage-gated, persistent, non-selective cation current in mouse (and guinea pig) vagal sensory neurons that is effectively inhibited by external divalent cations ≥100 µM but is insensitive to blockers of Na


Specifically, the Ca2+-ATPase transport activity was decreased in ER membranes isolated from cells lacking a +cta4 gene. Furthermore, a disruption of +cta4 resulted in 6-fold increase of intracellular Ca2+ levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca2+ pumping. These data provide compelling biochemical evidence for a P5A-type Cta4 ATPase as an essential component of Ca2+ transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast.This study establishes the role of P The calcium signal is terminated when cytosolic-free Ca2+ concentration is reduced to basal levels by Ca2+-ATPases and Ca2+/H+ exchangers that transport calcium out of the cell or sequester it in the organelles 2+ ions are required for the activities of numerous enzymes that catalyze the folding, modification, processing and trafficking of secretory proteins 2+ influx at the plasma membrane that serves to replenish the Ca2+-depleted organelle and to trigger calcium signaling pathways in both animal and yeast cells 2+/calmodulin-dependent protein phosphatase or calcineurin which is required for cytokinesis and ion homeostasis Calcium plays a key role in signal transduction in eukaryotic cells and modulates a variety of cellular functions. The calcium signaling is initiated by opening the Ca2+-ATPases which belong to the P2A subfamily of P-type ATPases 5A-type ATPases were localized to yeast ER, namely Cta4p ATPase of the fission yeast Schizosaccharomyces pombeSaccharomyces cerevisiae5A-ATPases are involved in calcium homeostasis in vitro in the presence of several cations including Ca2+, however, they failed to stimulate hydrolytic activity S. cerevisiae membranes S. cerevisiae Spf1p/Cod1p ATPase forms an oligomeric endomembrane complex 5A ATPase as a Ca2+-ATPase should be obtained from the measurements of ATP-dependent Ca2+ transport activity in native yeast membranes and comparison between wild type and null mutant cells. However, till now, there were no reports on Ca2+-ATPase activity in fission yeast membranes.In animal cells, the calcium transport to endoplasmic/sarcoplasmic reticulum is mediated by SERCA Ca5A-ATPases leads to sensitivity towards calcium stress 5A-type Cta4 ATPase in controlling ER Ca2+ store and signaling in S. pombe.Previous data show that the absence of ER located yeast PSchizosaccharomyces pombe used in this study were the wild type Fy1180 (+ otr1hR(SphI)::+ ura4-D18 leu1-32 ade6-ade6M210), the strain Hu185 expressing GFP-tagged Cta4p (+ cta4-GFP::kanMX6 otr1R(SphI)::ade6h+ura4-D18 leu1-32 ade6-M210), and the mutant strain Hu285 lacking Cta4p (− cta4::ura4+ ura4-D18 leu1-32 ade6-hM216) Strains of 2+ in yeast cells (cytosol and organelles) was measured as described 45CaCl2. After 5 h incubation at 30°C, cells were harvested rapidly by filtration onto GF/F filters (Whatman), washed with ice-cold buffer , placed in scintillation vials and processed for liquid scintillation counting on a liquid scintillation counter. The specific activity of the culture medium was determined in each experiment. Cell optical density was determined at 600 nm.Total cellular accumulation of CaTrichoderma (Sigma)/1 g of wet cells. After 50 min the incubation mixture of spheroplasts, old cells and cell walls was rapidly cooled and received EDTA, benzamidine and PMSF at 1.2 M sorbitol and 10 mM Tris–HCl, pH 7.4 to give final concentrations 1 mM of each protease inhibitor. The obtained mixture was added to a solution of 1.4 M sorbitol in 10 mM Tris-HCl, pH 7.4, centrifuged for 5 min at 3,000×g and then resuspended and homogenized in a Potter glass homogenizer using a lysis buffer . Total membranes were precipitated for 45 min at 120,000×g, resuspended in lysis buffer and loaded onto a 12-step gradient formed of 56, 52, 48, 45, 42, 39, 36, 33, 30, 25 and 20% sucrose (w/w) prepared in 10 mM MOPS-Na pH 7.2. The cocktail of the polypeptide protease inhibitors was applied to each step of gradient. After centrifugation for 2 h 45 min at 140,000×g membrane fractions were collected from the bottom and frozen. A 3-step sucrose gradient was formed of 50, 38 and 25% sucrose (w/w); membrane fractions enriched with vacuole, Golgi, ER/nuclear envelope were collected from the respective interfaces after centrifugation for 2 h 45 min at 140,000×gYeast membranes were isolated and fractionated according to Activities of organellar marker enzymes (NADPH cytochrome c oxidoreductase and GDPase) as well as the protein determination followed already published procedures 45Ca2+ uptake by isolated membranes vesicles was measured by the filtration method 2, 1 mM ATP, 9.8 µM EGTA, 10.4 µM CaCl2, 0.5 µCi 45Ca2+ (Amersham Life Sciences) and 2 µM cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP). At concentration of Cl− used in assay (160 mMKCl)) the membrane potential is converted into ΔpH from EMH+ or to ΔpCa2+ from EMCa2+. The reaction was initiated by adding 10–30 µL of suspension of total membrane vesicles or membrane fractions. After incubation at 30°C, 180 µL aliquots were injected into 10 mL of stop solution , filtered on 0.45 µm nitrocellulose filters (Millipore) and washed with 10 mL of the same buffer. Radioactivity retained on the filters was measured by scintillation counting. The specific activity of the reaction medium was determined in each experiment. The values were normalized per mg of total membrane protein.Yeast membranes from the sucrose gradient fractions (10 µL) and total membranes  were spotted on nitrocellulose membranes and probed with anti-BiP  or anti-GFP antibodies (1∶400). Anti-GFP antibodies were purchased from Sigma-Aldrich. Anti-BiP antibodies were provided by Prof. J. Armstrong . The blots were developed with peroxidase-conjugated secondary antibody .Spheroplasts were obtained and lysed as described above for membrane isolation. The homogenate was passed through a Sephadex G-25 column to remove the free phosphate. The solution eluted from the column was used in the calcineurin phosphatase activity assay. Calcineurin protein phosphatase activity was assayed using the Calcineurin Cellular Activity Assay Kit  according to the manufacturer's instruction. The assay was carried out in 50 µL volumes in a microtiter plate using RII phosphopeptide, the most efficient and commonly used peptide substrate for calcineurin. The reaction was started by adding the cellular extracts, followed by incubation at 30°C for 30 min, and terminated by adding the Malachite Green reagent according to the manufacturer's instruction. The activity of calcineurin protein phosphatase was determined by the amount of free phosphate released and normalized per mg of protein.2+ homeostasis, we investigated the effect of +cta4 deletion on cellular calcium levels. We found that mutant cells lacking Cta4 ATPase exhibited 6-fold increase in total calcium accumulation as compared to wild-type cells , indicating that remaining 50% of the transport was mediated by Ca2+-ATPases. Indeed, this ATP-dependent FCCP-insensitive 45Ca2+ transport was also sensitive to orthovanadate, the specific inhibitor of P-type ATPases, showing 85% and 98% inhibition at 0.5 mM and 1 mM, respectively (To examine whether Cta4p participates in Caectively .45Ca2+ transport (2+/H+ antiporters in mutant cells. 45Ca2+ transport mediated by Ca2+-ATPase (FCCP-insensitive transport) was 4-fold lower in cta4Δ membranes than in wild type (50∼0.2 mM (+cta4 is required for Ca2+-ATPase activity and Ca2+ sequestering in fission yeast membranes.The loss of Cta4p resulted in strong reduction (4.5-fold) of total ATP-dependent ransport  of whichild type  and 50% 0∼0.2 mM . These d2+ uptake, separation of the fission yeast membranes on a 12-step sucrose gradient was performed. Determination of sucrose density in addition to biochemical and immunochemical characterization showed that membrane fractions 1–11 were likely derived from the nucleus according to GFP-tagged Caf3-89 immunodetection  whereas most of this vanadate-sensitive activity disappeared in ER fraction of cta4Δ  of cta4Δ, it was augmented in vacuolar fractions  2+/CaM-dependent phosphatase calcineurin. Analysis of calcineurin specific activity in wild type and cta4Δ cells revealed that +cta4 deletion resulted in drastic, 10-fold stimulation in calcineurin activity compared to wild-type cells , and Pmr1p/Cps5p (SPBC31E1.02c) The members of P5A-type Cta4 ATPase in Ca2+ sequestration in the ER. Our conclusion follows from the finding that ATP-dependent, FCCP-insensitive and vanadate-sensitive Ca2+ transport activity was severely diminished in ER membranes isolated from cells lacking Cta4 ATPase. To our knowledge, this is also the first report describing a Ca2+-ATPase transport activity in fission yeast endoplasmic reticulum, Golgi and vacuole membranes. For evolutionary reasons that remain unclear, S. pombe and S. cerevisiae lack homologs of SERCA pumps. In this scenario, another ATPase should assure Ca2+ transport in ER. The present study provides biochemical evidence that the P5A Cta4 ATPase is essential for Ca2+-ATPase activity in ER and is crucial for establishing yeast ER homeostasis by regulating calcium transport. In our previous work only Ca2+/H+ exchange activity was measurable in membranes of S. pombe suggesting that Ca2+-ATPases were not expressed or not detected by unknown reasons 2+-transporters.Using biochemical and genetic approaches, this study establishes the role of P5A-ATPases led some authors propose that P5A-ATPases could be a phospholipid-ATPase mainly due to its homology with some flippases S. cerevisiae and other organisms does not support this alternative, since this enzymes shows a very low affinity for ATP . Elevated Ca2+ levels promote activation of calcineurin, essential for cta4Δ survival. In animal cells, the inhibitors of SERCA pumps activate the entry of Ca2+ into the cytoplasm through plasma membrane channels, a mechanism known as capacitative calcium entry or CCE 2+-ATPases also leads to increase of mRNA levels for the ER stress marker proteins BiP/GRP78 2+-ATPase Pmr1p 2+ influx through a plasma membrane HACS channel resulting in activation of calcineurin 2+ insensitive component of 45Ca2+ uptake was significantly increased in cta4Δ (unpublished data).We further showed that S. cerevisiae allowed the identification of group A mutants (HACS regulators) with spontaneous HACS activation and defects in endomembrane trafficking system, and group B mutants which require external stimuli (tunicamycin or α-factor) to activate Ca2+ influx. Importantly, our results place cta4Δ in group A, together with pmr1 mutant. Notably, spf1 and pmc1 mutants belong to group B. It should be emphasized that loss of Spf1/Cod1 ATPase in S. cerevisiae did not result in changes in cellular calcium levels 2+ influx and HACS +cta4 disruption. In this respect, it is noteworthy that mutant lacking Cta4 ATPase was sensitive to inhibition of calcineurin cta4Δ cells undergoing ER stress. This finding is in agreement with the demonstration of requirement of calcineurin for a survival of mutants with activated HACS S. pombe. However, one of these targets has been already identified. We showed that cta4Δ mutant displays an increase in Ca2+-ATPase activity in vacuole membranes as compared to wild-type. Thus, induced calcium sequestration to vacuoles possibly compensates for the loss of the Cta4p from the ER, and contributes to lowering the cytoplasmic calcium. Also, we found that cta4Δ exhibits a decrease in activity of Ca2+/H+ antiporters. Our findings support the idea that elevated intracellular calcium in cta4Δ leads to activation of the calcineurin which, in turn, differentially regulates other Ca2+ transporters. This is consistent with results obtained for the S. cerevisiae Vcx1, a vacuolar H+/Ca2+ exchanger, which activity is inhibited by calcineurin 2+ in calcineurin-dependent manner S. cerevisiae Golgi-localized Ca2+-ATPase Pmr1p was shown to be dependent on calcineurin S. pombe, Pmr1p/Csp5p is localized to ER cta4Δ. Our data suggest that vacuolar Ca2+-ATPase Pmc1p assumes a leading role in calcium homeostasis in cta4Δ genetic background. This suggests that vacuolar pump could serve as a backup system when Cta4 ATPase is missing and under calcium overload. Indeed, the S. pombe pmc1+ was required for growth in medium in the presence of high extracellular CaCl2cta4Δ. This supposition is consistent with the demonstration of the induction of PMC1 transcription in S. cerevisiae mutants lacking Spf1p/Cod1p P5AATPase using ß-galactosidase reporter plasmid It has been suggested recently that the activation of HACS is a major response to defects in the secretory, endosomal and vacuolar protein trafficking pathways cta4Δ mutant is under ER stress as revealed by the increased expression of the ER stress indicator, the chaperone BiP. The distribution of BiP in Golgi and vacuole membrane fractions isolated from cta4Δ mutant deserves special mention. Disruption of +cta4 may affect not only BiP distribution but also that of other proteins of the secretory pathway. In agreement, it was demonstrated that loss of another P5A ATPase, Spf1/Cod1p from S. cerevisiae, resulted in altered distribution of another ER membrane protein, Sec12 5A ATPase gene MIA from Arabidopsis resulted in major changes in expression of genes involved in protein secretion 5A ATPases is to control the proper functioning of secretory pathway and protein targeting. This finding is not without precedence, since deletion of S. cerevisiae Pmr1 Ca2+-ATPase also resulted in ER stress The data also established a critical role of Cta4p in maintaining the ER folding environment. The findings reported here indicate that +cta4 resulted in ER stress and inability to cope with accumulation of unfolded proteins. Although the ER-associated protein degradation constitutes a main mechanism for elimination of unfolded protein, there is growing evidence for a vacuole participation in this process. A recent study in tobacco has shown that BiP is transported to the vacuole and that the ER export of BiP occurs via COPII-dependent transport to the Golgi apparatus cta4Δ vacuolar disposal of proteins could act in addition to ER-associated protein degradation to improve the efficiency of quality control throughout in the secretory pathway. The stimulation of vacuole Ca2+ transport observed in cta4Δ might be required to maximize vacuole functioning, besides of lowering cytoplasmic calcium levels. Thus, the vacuole shares a role in calcium homeostasis and quality control with ER.Loss of 2+ or, at least, regulates Ca2+ transport in ER by influencing functional localization of Pmr1p in the ER. Although both alternatives remain to be investigated, the last one should not be the case for Spf1/Cod1p from S. cerevisiae since Pmr1p is localized to Golgi in this yeast Our results adds biochemical substantiation to the argument that Cta4p might transport Ca2+ transport mediated by Ca2+-ATPase in fission yeast membranes. Our findings depict a crucial role of P5A-type Cta4 ATPase in Ca2+ transport in ER and regulation of Ca2+ influx system. Finally, our data provide a platform for future studies of the signaling network which encompasses the calcineurin and Ca2+-ATPases activity and controls the calcium homeostasis and the function of the endomembrane system in S. pombe.In conclusion, we provide functional evidence of Ca


GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca2+-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca2+-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca2+-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene  Inherited retinal diseases are caused by a heterogeneous group of mutations in retinal genes . Among t2+, which binds to and dissociates from Ca2+-sensor proteins under oscillating changes in cytoplasmic Ca2+-concentration [Ca2+]. A multi-protein complex operating at the interface between changing concentrations of cGMP and [Ca2+] consists of a sensory type membrane-bound guanylate cyclase that is regulated by guanylate cyclase-activating proteins   . PreviouROS-GC1) .2+-sensitive control switch module . The mutation P575L was found in a family, where clinically affected members suffer from progressive cone degeneration  exactly as described previously . DNA was6 cells were harvested in 100 μL of a buffer containing 5 mM KCl, 20 mM MgCl2, and 150 mM phosphate buffer pH 7.2. For transfection, 5 μg of DNA was added and cells were electroporated with the CLB system (Lonza). Further treatment of cells and harvesting for subsequent guanylate cyclase assays was done exactly as described . In a second set of guanylate cyclase assays we incubated recombinant ROS-GC1 with increasing concentrations of GCAP1 and GCAP2 (0–20 μM) for 30 min under Ca2+-free conditions. GCAPs were liganded with Mg2+  of GCAPs for WT and mutant ROS-GC1. Activity values were obtained from at least 3–4 different data sets and were used to calculate the mean ± standard deviation (SD). A student’s t-test was performed to check for significant differences between two sets of data.Recombinant human ROS-GC1 in HEK cell membranes was measured as described before . In addiications . Data weith Mg2+  using thith Mg2+ . The lat+/K+-ATPase , sc-48345, mouse monoclonal IgG2b; Santa Cruz Biotechnology), and anti-calnexin . Secondary antibodies were donkey anti-rabbit conjugated to Fura350 (dilution 1:200) from Invitrogen and goat anti-mouse conjugated with Dylight594 from Thermo Scientific, USA used in a dilution of 1:500. Incubation and washing buffers were exactly as described  and using specific markers for the plasma membrane  and for the endoplasmic reticulum  we obtained a staining pattern in the overlay image  in agreement with published results . Activities in the presence of GCAP1 and 2 at saturating [Ca2+] revealed almost no difference between P575L and WT. However, a difference was observed, when GCAP1 and 2 were incubated with ROS-GC1 at low [Ca2+]  resulting in a 25–30% decrease of maximal activity. In sharp contrast to P575L, the mutant P1069R did not exhibit any activity, neither in the presence nor in the absence of Ca2+, EGTA, or GCAPs (data not shown). We also tested activities of cells transiently expressing the mutant or a stably transfected cell line. Since the cellular localization of this mutant appeared normal and was very similar to the active WT and P575L forms , we conclude that the lack of GC activity was a consequence of an impaired catalytic domain.The mutant P575L exhibited similar basal GC activities as WT  and Figure 4B (+GCAP2), a summary of the IC50 values (mean ± SD) is displayed in Table 1. For both Ca2+-sensor proteins we observed a similar two-fold shift of the IC50 values to lower [Ca2+], in the presence of GCAP1 from 0.529 (WT) to 0.270 μM (P575L) and in the presence of GCAP2 from 0.269 μ(WT) to 0.128 μM (P575L).The key feature among ROS-GC1 properties is the Ca50 values had previously been reported for some autosomal cone dystrophy related mutations that are located in a so-called “mutation hotspot” region within or near the residue R838 in the dimerization domain . GCAP1 and 2 were Ca2+-free, but they had Mg2+-bound (50-values differed only minimally between WT and the P575L mutant and overlapped within the measured standard deviations (Table 2+-sensitivity of the P575L mutant.A shift in the ICn domain . One reaof GCAPs . We test2+-bound  under thThe single point mutation P575L is located in direct C-terminal neighborhood of a small stretch of 20 amino acids, which was previously identified as an essential regulatory and/ or interaction site in bovine ROS-GC1 for GCAP1 (L559–I578 corresponding to peptide #34a in in vitro assays.The position H1019P is located in the short β6-sheet of the CCD in close vicinity of the β5-sheet that harbors amino acids R995, C997, L998, and F999, which are critical for binding the purine ring of the GTP substrate . ReplaceIn our test system the mutation P1069R had the most dramatic consequences leading to a completely inactive enzyme. This position is sandwiched between the binding sites of GCAP2 and S100β  and we c2+-sensitive cyclase activation profile, it seems to have long-term effects in the distortion of the Ca2+-cGMP homeostasis triggering apoptotic pathways. Similar consequences have been discussed for GCAP1 mutations correlating with certain forms of cone-rod dystrophies (Inherited retinal disorders can affect visual function in different manners. In the present study we investigated the biochemical consequences of three point mutations in the GUCY2D gene, which cause different forms of visual impairment. The mutation P575L causes a progressive form of cone degeneration in affected patients, which very often correlates with symptoms of photophobia, decrease of central vision and impairment of color vision. Since the mutation did not lead to a complete loss of guanylate cyclase function, cone cells expressing the mutated ROS-GC1 protein very likely maintain key features of photoreceptor physiology. But since the mutation induces a dysregulation of the Catrophies . The othtrophies . Clinicatrophies . The cGMThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cancer chemopreventive response to PEITC is characterized by its ability to inhibit multiple oncogenic signaling pathways, including nuclear factor-κB, Akt, and androgen receptor. The present study demonstrates, for the first time, that PEITC treatment activates Notch signaling in malignant as well as normal human prostate cells. Exposure of human prostate cancer cells  and a normal human prostate epithelial cell line (PrEC) to PEITC resulted in cleavage (active form) of Notch1 and Notch2, and increased transcriptional activity of Notch. In PC-3 and LNCaP cells, PEITC treatment caused induction of Notch ligands Jagged1 and Jagged2 (PC-3), overexpression of γ-secretase complex components Presenilin1 and Nicastrin (PC-3), nuclear enrichment of cleaved Notch2, and/or up-regulation of Notch1, Notch2, Jagged1, and/or Jagged2 mRNA. PEITC-induced apoptosis in LNCaP and PC-3 cells was significantly attenuated by RNA interference of Notch2, but not by pharmacological inhibition of Notch1. Inhibition of PC-3 and LNCaP cell migration resulting from PEITC exposure was significantly augmented by knockdown of Notch2 protein as well as pharmacological inhibition of Notch1 activation. Nuclear expression of cleaved Notch2 protein was significantly higher in PC-3 xenografts from PEITC-treated mice and dorsolateral prostates from PEITC-fed TRAMP mice compared with respective control. Because Notch signaling is implicated in epithelial-mesenchymal transition and metastasis, the present study suggests that anti-metastatic effect of PEITC may be augmented by a combination regimen involving a Notch inhibitor.Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive component of edible cruciferous vegetables with  In vivo chemopreventive efficacy of PEITC against prostate cancer has now been established in a transgenic mouse model  e.g., lung, colon, and esophagus) by this dietary component has also been documented N-acetylcysteine conjugate in vivo in prostate cancer xenografts Practical and safe modalities for chemoprevention of prostate cancer are clinically attractive because of high mortality associated with this malignancy in American men Safety, bioavailability, selectivity towards cancer cells, and ability to target multiple oncogenic pathways are desirable attributes of a clinically useful cancer chemopreventive agent. Research thus far indicates that PEITC meets all these criteria. Firstly, PEITC is well-tolerated by experimental rodents The present study extends these observations e.g., Jagged1 and Jagged2), which is thought to induce a conformational change within the Notch receptor resulting in exposure of an S2 cleavage site for tumor necrosis factor-α converting enzyme Ligand-dependent activation of Notch is complex requiring cleavage by γ-secretase complex in vivo growth in xenograft model, and cell migration. We questioned if differential response of LNCaP versus PC-3 cells to PEITC-mediated alterations in Notch signaling components was related to androgen-independent phenotype. We addressed this question using an androgen-independent variant of LNCaP cells (LNCaP−C4-2). Response of LNCaP−C4-2 cells to PEITC-mediated changes in Notch signaling proteins was generally similar to that observed in the parental LNCaP cells  to determine if PEITC-mediated activation of Notch1 and Notch2 was unique to cancerous prostate cells. This was a worthy research objective considering striking differences have been noted with regards to effect of PEITC between cancerous and normal prostate cells. For example, we have shown previously that the PrEC cell line is significantly resistant to PEITC-mediated inhibition of oxidative phosphorylation, reactive oxygen species generation, and apoptosis induction compared with PC-3 and LNCaP cells Because the effect of PEITC treatment was relatively more pronounced and sustained on Notch2 cleavage compared with cleaved Notch1, we proceeded to determine cleaved Notch2 levels in DMSO-treated control and PEITC-treated LNCaP and PC-3 cells. PEITC-treated LNCaP and PC-3 cells exhibited a marked increase in the nuclear levels of cleaved Notch2 in comparison with DMSO-treated control . These rNotch1, Notch2, Jagged1, and Jagged2 are shown in Notch1 (2.5 and 5 µM PEITC) and Jagged1 (5 µM PEITC) mRNA was increased significantly upon 8-hour treatment of LNCaP cells with PEITC (Notch2 (5 µM PEITC) and Jagged2 (5 µM PEITC) mRNA levels in LNCaP cells ]-S-phenylglycine-t-butyl ester; hereafter abbreviated as DAPT} to further test the role of Notch in PEITC-induced apoptosis. PEITC-mediated increase in levels of cleaved Notch1 protein, but not cleaved Notch2, was markedly suppressed by co-treatment with 50 µM DAPT . As expeBecause Notch signaling is implicated in cell invasion and epithelial-mesenchymal transition (EMT) DAPT alone caused a modest decrease in LNCaP  and PC-3in vivo relevance of the cellular findings  is an androgen receptor selective co-repressor LNCaP and PC-3 cells exhibit striking differences with regards to PEITC-mediated alterations in Notch signaling components, especially Notch ligands and γ-secretase components. Two obvious possibilities deserve attention to explain these differences in PEITC response between androgen-responsive LNCaP cells (wild-type p53) Activation of Notch2 by overexpression of its intracellular domain promotes apoptosis in MDA-MB-231 cells Metastasis is the primary cause of mortality from prostate cancer. Notably, Notch has been implicated in EMT In conclusion, the present study demonstrates that PEITC treatment promotes cleavage of Notch1 and Notch2 leading to their transcriptional activation in both cancerous  and normal (PrEC) prostate cells. PEITC-induced apoptosis in prostate cancer cells is modestly attenuated by knockdown of Notch2, but not by pharmacological inhibition of Notch1 activation. On the other hand, targeted depletion of Notch2 by siRNA as well as inhibition of Notch1 activation using DAPT augments prostate cancer cell motility inhibition by PEITC.in vivo studies We used archived tissue sections from our previously published PEITC (purity >98%) was purchased from LKT Laboratories . Cell culture reagents were purchased from Invitrogen-Life Technologies . Antibodies used in the present study were purchased from the following sources: antibodies against cleaved Notch1, Presenilin1, Jagged1, Jagged2, and Nicastrin were from Cell Signaling Technology ; an antibody specific for detection of cleaved Notch2 was from EMD-Millipore ; and anti-actin antibody was from Sigma-Aldrich . A γ-secretase inhibitor (DAPT) was purchased from Calbiochem . Notch2-targeted siRNA was purchased from Santa Cruz Biotechnology . A non-specific control siRNA was purchased from Qiagen .LNCaP, PC-3, and DU145 human prostate cancer cell lines were obtained from American Type Culture Collection , and maintained as described by us previously Stock solution of PEITC was prepared in DMSO and an equal volume of DMSO  was added to controls. After treatment with DMSO or PEITC, cells were collected and processed for immunoblotting as described by us previously Cignal RBP-Jk luciferase reporter kit from SABiosciences-Qiagen was used to determine the effect of PEITC treatment on transcriptional activity of Notch. The RBP-Jk [C protein binding factor 1/Suppressor of Hairless/Lag1 (CBF1/Su (H)/Lag 1)] is a direct downstream modulator of Notch signaling 5) were grown on coverslips in 12-well plates and allowed to attach by overnight incubation. Cells were treated with DMSO (control) or PEITC (5 µM) for 8 hours, fixed with 2% paraformaldehyde for 1 hour at room temperature, and permeabilized using 0.5% Triton X-100 for 5 minutes. Cells were then incubated with phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin and 0.15% glycine for 1 hour followed by overnight incubation with anti-cleaved Notch2 antibody (1∶500 dilution in above buffer) at 4°C. Next day, cells were treated with 2 µg/mL of Alexa Fluor-488-conjugated secondary antibody for 1 hour at room temperature. After washing with PBS, cells were treated with 50 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature to stain nuclear DNA. Cells were mounted and visualized under a fluorescence microscope.LNCaP or PC-3 cells  with 55°C annealing (45 seconds for Notch1 and Notch2), 53°C annealing (45 seconds for Jagged1 and Jagged2) for 40 cycles. Relative gene expression was calculated using the method described by Livak and Schmittgen and normalized against GAPDHTotal RNA from DMSO-treated control and PEITC-treated cells was isolated using RNeasy kit (Qiagen). First-strand cDNA was synthesized using Superscript reverse transcriptase (Invitrogen-Life Technologies) with oligo (dT)LNCaP or PC-3 cells were transfected at ∼50% confluency with 100 nmol/L of Notch2- targeted siRNA or control siRNA using Oligofectamine. Twenty-four hours after transfection, cells were treated with DMSO (control) or PEITC (5 µM) for 24 hours. Subsequently, cells were collected and processed for western blotting, quantitation of histone-associated DNA fragment release into the cytosol (a method for quantitation of apoptosis), and cell migration. Histone-associated DNA fragment release into the cytosol was quantified using Cell Death Detection ELISA kit  by following the manufacturer's instructions.Transwell Boyden chambers containing 8.0 µm pore size polycarbonate filter  were used to determine cell migration. LNCaP or PC-3 cells were transfected with a control siRNA or Notch2-targeted siRNA as above, and used for cell migration assay described by us previously Immunohistochemistry was performed essentially as described by us previously for other proteins