T leukemogenesis is a multistep process, where the genetic errors during T cell maturation cause the healthy progenitor to convert into the leukemic precursor that lost its ability to differentiate but possesses high potential for proliferation, self-renewal, and migration. A new misdirecting “leukemogenic” signaling network appears, composed by three types of participants which are encoded by (1) genes implicated in determined stages of T cell development but deregulated by translocations or mutations, (2) genes which normally do not participate in T cell development but are upregulated, and (3) nondifferentially expressed genes which become highly interconnected with genes expressed differentially. It appears that each of three groups may contain genes coding ion channels. In T cells, ion channels are implicated in regulation of cell cycle progression, differentiation, activation, migration, and cell death. In the present review we are going to reveal a relationship between different genetic defects, which drive the T cell neoplasias, with calcium signaling and ion channels. We suggest that changes in regulation of various ion channels in different types of the T leukemias may provide the intracellular ion microenvironment favorable to maintain self-renewal capacity, arrest differentiation, induce proliferation, and enhance motility. T cell acute lymphoblastic leukemias (T-ALL) are aggressive neoplastic disorders of the lymphoblasts committed to the T lineage. T-ALL accounts for 15% of pediatric and 25% of adult ALL cases i represent ubiquitous signaling mechanism that regulates different phases of T cell physiology, including the proliferation, lineage decision in the maturation, and antigen activation [2+]i in resting cell or generate Ca2+ signal of a particular frequency and amplitude, time course, and intracellular location, every cell possesses the unique set of components, involved in this regulation, a so-called Ca2+ toolkit i have been detected as the cell grows and passes through G1, G1/S, and mitosis. Ca2+ not only operates upstream of the cell-cycle machinery by regulating the expression, activity, and/or location of the transcription factors that control expression of the G1 cyclins , and NFAT), but also acts more directly on the cyclins, CDKs, and/or their small protein inhibitors to regulate the assembly and activation of CDK complexes .2+]i raise, subsequent events related to a new gene expression all require a sustained Ca2+ influx to maintain [Ca2+]i at level higher than basal one in resting cells. In addition to CRAC channels, other Ca2+-permeable channels have been found in T lymphocytes and proposed as Ca2+ influx pathways during different physiological events (as discussed below).Whereas initial T cell activation is related to short-term local i is an indispensable factor to maintain the Cn activity, since mutations resulting in a persistent Cn activation, independent of Ca2+, were never reported.Increased Cn/NFAT activity by the Notch signaling was shown to involve a downregulation of calcipressin through the Hes1-dependent mechanism . As far ll lines , 112. In to Ca2+ . But stiAs was already mentioned earlier, the NFAT activation is a hallmark of the T-ALL. While activated mutations of NFAT genes have been never observed in human cancers, the aberrant NFAT signaling in tumors was suggested to involve either its overexpression and/or hyperactivity (reviewed in ). NFAT h2+ rise generation in T lymphocyte. Less considered pathway is related to a nonneuronal lymphoid cholinergic system. Lymphocytes possess all components of independent cholinergic system that include the acetylcholine (ACh), choline acetyltransferase (ChAT), its synthesizing enzyme, and both muscarinic  and nicotinic (nAChR) ACh receptors [2+]i with Ca2+ oscillation via the IP3-evoked Ca2+ rise from the intracellular store. This pathway was suggested as an amplification mechanism to increase the IL2 and IL2R production [+ cells into the CTL [2+ influx in leukemic cells may be maintained by elevated Ach production and autocrine stimulation through the mAChRs ligation.Calcium mobilization, following the TCR ligation during the T cell activation is essential but is not the only way of the Caeceptors –116. Humoduction  and the  the CTL . Emergin the CTL . As for  the CTL . It coulTaking into account the purpose of the present review, we have undertaken the bibliographic search of the data on possible involvement of transcriptional elements, misregulated in the T-ALL, in the ion channel expression and activity.+ cell line as a model [To identify TAL1 direct target genes, Palomero and colleagues have undertaken the chromatin immunoprecipitation experiments with antibodies raised against the TAL1 in Jurkat CD4 a model . It was 2+-permeable channels in primary samples and cell lines derived from GBM. Functionally, TRPC6 caused a sustained elevation of the intracellular Ca2+ coupled to the activation of the calcineurin-related NFAT pathway. TRPC6 was shown to be required for the development of the aggressive tumor phenotype, because a knockdown of the TRPC6 inhibited the glioma growth, invasion, and angiogenesis. Notch-dependent transcription of the TRPC6 was reported in pheochromocytoma PC12 cells [Notch 1 activation, being a hallmark of many types of the T-ALL, is also involved in the pathophysiology of other cancers. Then we undertook a search for data on the possible relationship between the Notch1 activation and ion channel expression. For example, aggressive and malignant state of glioblastoma multiforme (GBM), the most frequent and incurable type of the brain tumor of adults, was shown to be related to an increased activation of the Notch1 provoked by hypoxia . Notch1 12 cells . Interes12 cells .2+-binding protein CHGA, and peptide hydrolase MEPIA were pointed out among the non-DE genes in T-ALL.Traditionally, the T-ALL diagnostics and corresponding therapeutic strategies are based on the differential expression (DE) of genes, that occurred in the T-ALL patients as compared to healthy persons. However, as it was recently pointed out by Maiorov and colleagues (2013), the expression of some genes might not vary in the T-ALL, but, instead, these genes may be interconnected with highly differentially expressed ones . They ha+-selective, Ca2+-selective, and nonselective cation and anion channels. Wherein, their local combinations one with another and with other signaling components form a highly specific microenvironment, essential for cellular performance. The first purpose of this chapter is to give an overview of ion channels, expressed in T-cells, along with their known functions and giving emphasis to those, which are differentially expressed in healthy and leukemic cells. The second purpose is to revise the channel-to-channel functional communications, especially those related to a formation of specific Ca2+ signal. The third is to present, whenever it is possible, the structural and functional view on membrane signaling complexes, involving an ion channel as a core element.Tumor cells survival and proliferation, activation, differentiation and malignant progression, invasiveness/migration , and, at last but not the least, the resistance to anticancer therapies, all these critically depend on the function of ion channels. T lymphocytes and leukemic T cells bear authentic orchestras of K+-) selective channels reported up to the date in healthy or neoplastic lymphoid cells belong to several families: voltage-gated (Kv), Ca2+-activated (KCa), and tandem-pore domain (K2P) channels. K+ channels may act via their ionotropic function or via noncanonical nonconducting mechanisms, that is, via direct interaction with other membrane or cytosolic proteins. Channels' mediated K+ transport causes a change of membrane voltage, affects a driving force for Ca2+ influx, and, via changes of intracellular K+, regulates the cell volume  and, more specifically, affects the activity of the intracellular machinery, for example, activation of caspases in the course of apoptosis [+ conduction), the G2/M one occurs with a depolarization and low K+ conductance [+ channels cause G1 arrest [+ efflux via voltage-gated  K+ channels [2+ bursts. The former includes activation of CDKs and culminates through phosphorylation of Rb1 in activation of E2F transcription factors. During progression of the G1 phase several processes like expression of AP1 (JUN and FOS) and CREB transcription factors, as well as regulation of cyclins, are calmodulin dependent, whereas transport of the transcription factor NFAT to nucleus requires the calcineurin-mediated dephosphorylation [2+ signaling is critically dependent on the activity of partner K+ channels as will be discussed below.Potassium-  only a tiny fraction of Kv1.3 channels remains open at a steady state [+ equilibrium, which are observed at the G1/S transition in the cell cycle, require the activity of some voltage-independent K+  channel(s), open at this voltage range. Kv1.3 channel at its C-terminus forms a functional complex with the β1-integrin, the PDZ-domain protein SAP97, linked to the p56Lck kinase and an adapter ZIP protein; the N-terminus in turn could bind a Kvβ2 subunit (redox sensing), which may eventually interact with ZIP protein [v1.3 and β1-integrin moieties and activates the integrin function, adhesion, and migration, whereas specific Kv1.3 channel blockage prevents the integrin signaling [v1.3 in T cells include but not restricted to (1) control of membrane potential against a depolarization challenge, depending on the T cells subset [2+ influx by CRAC, generation of Ca2+ oscillations (together with KCa3.1 and TRPM4). A sustained, lasting over hours, Ca2+ increase, which is primordial for new gene expression, is triggered in T cells via the Ca2+/calcineurin/NFAT pathway [v1.3 and KCa3.1 tends to abolish Ca2+ oscillations, with an impact on T cells proliferation [v1.3 abolished oscillations of the CRAC-mediated Ca2+ entry but not the average Ca2+ entry [Ca channels activity (see below). Obviously, CRAC and KCa3.1 activities are linked in a feedforward manner, so that the activation of KCa3.1 by Ca2+ influx via CRAC will hyperpolarize the membrane, increasing CRAC-mediated Ca2+ influx, and so forth. To reverse this Ca2+ increase, additional mechanisms are required which may be CRAC inactivation by Ca2+ [2+-impermeable member of the TRP family, the TRPM4 (see below). Kv1.3 is a predominant  K+ channel in naïve human T cells, which functionally express just few KCa3.1. Kv1.3 plays a very essential role in the lymphocyte activation and associated Ca2+ signaling and IL-2 production. Upon the activation, differential behavior is observed in diverse T cell subsets. In activated TEM (effector memory) cells Kv1.3 is selectively upregulated (highKv1.3 : lowKCa3.1 phenotype); on the contrary, in TCM  cells, Kv1.3 only modestly upregulated, whereas KCa3.1 dramatically (by 1.5 order of magnitude increase), producing lowKv1.3 : highKCa3.1 phenotype [v1.3 decreases the proliferation of TEM without significant effect on TCM and naïve cell population [2+ influx via CRAC. Selective Kv1.3 suppression was only efficient upon Ca2+-dependent lymphocyte activation and not in the cases of CD28 or IL-2-induced activation, which is independent on the intracellular Ca2+ rise [v1.3-mediated cells proliferation was unaffected in a poreless mutant  but abolished in a mutant with altered voltage gating [v1.3 protein upon the channel opening may be sufficient for an efficient signaling, without involving a K+ flux or membrane polarization. Such conformational coupling may be mediated by a close association of Kv1.3 channels with β1-integrin [K mammals , which iaddition . Kv1.3 idy state –139. Thuignaling . Functios subset , 143; . KCNE 4 but not 2 physically interacts and substantially modifies the Kv1.3 gating, decreases the channel surface expression, and impairs channel's targeting to lipid rafts [Comparison of the K T cells , 159–163 T cells , 164–167per cell . Taking involved  one may k kinase  as well k kinase  is preserivation . Healthyid rafts . Upon Juid rafts . Voltageid rafts  and Jurkid rafts , suggest+ channels have dual expression, in the plasma and intracellular membranes. Kv1.3 channel, discussed above, is expressed both in plasma and inner mitochondrial membranes. Plasma membrane Kv1.3 may be selectively blocked by membrane-impermeable toxins, whereas mitochondrial Kv1.3 is only blocked by membrane-permeable drugs; blockage in the latter case induced apoptosis in cancer cells, including leukemic ones [v1.3 interacts directly with Bax in Jurkat cells. This gives rise to a sequence of events, including the hyperpolarization of the inner mitochondrial membrane, ROS generation, and cytochrome c release from mitochondria, thus, promoting the intrinsic apoptotic pathway [Here we will mainly discuss the roles, played by plasma membrane ion channels. However, several intracellular channels and transporters, expressed in nuclear, ER, and mitochondrial membranes, especially the latter, play important roles in cancer cells proliferation and survival . In partmic ones . In addi pathway .Kr) K+ current, activating at membrane depolarization and mediating action potential repolarization. It has very peculiar biophysical properties, possessing a rapid inactivation at positive potentials and a rapid relief of inactivation at negative ones. Therefore, the steady-state I/V relationship for this channel is bell-shaped, with a peak around −10 mV, where also the channel's open probability reached its maximum. Yet, channel's deactivation  at negative potentials is slow (hundreds of milliseconds). Thus, at the peak and plateau of the action potential hERG1 conductance is greatly reduced due to the inactivation; it started to increase and reached its maximum at phase 3 repolarization [+ efflux, promoting further repolarization. However, the midpoint of the hERG1 activation is around −30 mV, so that it possesses a significant activity down to −40 mV, which is close to resting potential in some nonexcitable cells, including lymphocytes. hERG1 is undetectable or expressed at very low levels in healthy immune cells. But it is frequently abnormally overexpressed in many cancer types, in particular, in acute lymphoblastic as well as in chronic lymphocytic (CLL) and both in acute and chronic myeloid (AML and CML) leukemias [+ cells from peripheral blood, but only after cytokine stimulation [β1-integrin, or even in triple protein complexes  channels are normally expressed in excitable cells, primarily, in neurons and heart . In hearrization . In conceukemias , 176–178eukemias  and alsoomplexes , thus limembrane . Cancer poptosis . Althougpoptosis . In hearpoptosis , hERG1 fCa3.1 (IKCa1), intermediate conductance KCa channel, is voltage independent and is activated by Ca2+-CaM (CaM bound to C-terminus) with an apparent Kd = 300 nM [Ca3.1 also requires the phosphatidylinositol-3 (PI(3)P) for its activity, albeit it is not clear how this activation achieved; the effect on the channel is indirect [Ca3.1 current and efficiently modulates, in opposite way by NDPK-B or PHPT, Ca2+ influx and CD4+ T cells proliferation after stimulation [Ca provokes a stable hyperpolarization down to equilibrium potential for K+, EK [2+ channels (see below), where CRAC (SOCE) is believed to be a central Ca2+ influx channel. Although resting T cells express only few copies of KCa3.1, it is rapidly upregulated upon the activation and its expression level rises about 25-fold [Ca3.1 channel is involved in various cellular processes, namely, activation via TCR-engagement and stabilization of the immunological synapse; control of membrane potential, RVD, cell migration, and tumor-related angiogenesis [Ca3.1 interacts functionally with Kv1.3 and CRAC [K= 300 nM . KCa3.1 indirect . Downstrmulation , 187. Thr K+, EK . This hy 25-fold . KCa3.1 ogenesis , 190. KCand CRAC , 190.Ca2.2 (SKCa2), small conductance KCa channel, is expressed in a variety of healthy tissues, including brain and muscle [Ca3.1 expression in activated healthy T cells [Ca2.2 is activated by Ca2+ with an apparent Kd = 700 nM and is upregulated by p56Lck [0/G1 compared to G2/M phase, supporting a sustained Ca2+ influx [Ca2.2 silencing but not Kv1.3 inhibition suppresses the Ca2+ entry; it can be restored by the ectopic expression of KCa3.1 [Ca2.2 and KCa3.1 may be interchangeable, but it is not clear to which extent. It should be noted that in Jurkat the functional expression of KCa2.2 channels is constitutively high and can be compared to the KCa3.1 expression in activated healthy T cells [Ca2.2 expression both at mRNA and functional expression in the membrane levels [Kd muscle . It was  T cells , 193. KCy p56Lck . It has + influx . In Jurk T cells , 193. Ree levels .+ selective channels, genetically unrelated to the former two families (KCa and KV). Each channel subunit possesses two pore domains, and a minimal number of transmembrane segments, two per pore, as compared to the 6-TM subunit structure of KCa and KV. Consequently, functional K2P channels are dimers; functional heterodimers were reported only for closely related TASK1 and TASK3, and the rest of active K2P channels are homodimers. There are 14 members of K2P family discovered in mammals  and then completely inactivated in a half of hour, causing a strong membrane depolarization, whereas the Kv1.3 contribution to the K+ efflux was more constant in time [2+-dependent  Ca2+ channels of the plasma membrane. In lymphocytes, contrary to this, Ca2+ rise in cytosol is mediated by the store-operated Ca2+ entry (SOCE), named also CRAC, for Ca2+ release-activated Ca2+ current, that is, Ca2+-selective current of the plasma membrane, activated by the Ca2+ depletion in the ER. It is extremely (factor > 1000) selective for Ca2+ over monovalent cations and has extremely low single channel conductance for Ca2+ (30 fS), which is compensated by a very high channels density (ca. 100 channels/μm2). Due to its intrinsic inward rectification, Ca2+ influx via CRAC is strongly potentiated by membrane hyperpolarization, whereas depolarization reduces Ca2+ entry in lymphocytes [2+-selective CRAC, but now there is also an ample evidence that TRPCs and CRAC are functionally and physically separated [2+ replete stores STIM proteins are randomly distributed at the membrane surface, and store depletion causes oligomerization of STIM in special contact areas with plasma membrane, where cytosolically exposed STIM domain directly interacted with both N- and C termini of Orai1, thus, causing CRAC activation  [Caphocytes . For a leparated . Crucialeparated . Orai doeparated . Store dlls (see  and refells (see . Relativfunction . As CRACing Ca2+  and membs (2014) .2+-permeable ones; they resemble by transmembrane topology but distantly are related to voltage-gated  channels [TRP channels  are cationic, mostly nonselective and Cachannels , 214. Thchannels .Ca/PNa = 1–5), with linear or dual-rectifying I/V relation [2+-binding sites, a EF-hand and CIRB, a calmodulin/IP3R-binding domain and may be directly regulated by intracellular Ca2+ [2+/CaM pathway [trpc2 is a pseudogene in humans) subgroups. A common property of the latter subgroup is their activation by DAG (or its membrane permeable form OAG), whereas TRPC1/4/5, albeit being dependent on the PLC activity, are not responding to DAG [2+, Ba2+, and Sr2+ into Jurkat and PBLs; on the contrary, in case of SOCE (CRAC) activation cation influx was Ca2+-selective, without measurable Ba2+ component [2+ influx, induced in HPB-ALL cells by Δ9-tetrahydrocannabinol, was less sensitive to SOCE and, based on pharmacological evidence, was mediated by TRPC1 via CBD-2 receptors and DAG [2+ influx and Ca2+-dependent NO production [+ caused a reduction of intracellular Ca2+ only under limiting external Ca2+ (<0.1 mM) and slightly (<20%) decreased the proliferation of activated cells [2+ entry induced by anti-CD3 antibodies in Jurkat cells [2+ signaling.TRPC 1, 3, and 6 were reported in PBL and Jurkat cells, Jurkat expresses additionally TRPC 4 and 5 . All aborelation . TRPC chlar Ca2+ . Mostly, pathway . Accordig to DAG . OAG-actomponent . In HPB- and DAG . Yet the and DAG  as well  and DAG ; in the  and DAG . Importaoduction . It remaed cells . Howeverat cells , confirm+, Na+, and Ca2+ [2+ [+ precursors and in hematologic malignant cell lines of different lineages, like Jurkat cells (T-ALL), K562 (erythromyeloblastoid leukemia), AML-193 (acute monocytic leukemia), U937-ecoR (monocytic leukemia), and TF-1 (erythroleukemia) [2+ influx varied depending on the method of stimulation. In Jurkat cells, activation by ConA led to late Ca2+ influx via TRPM2 without CRAC involvement, whereas TRPC (TRPC3?) channels were responsible for anti-CD3-activated Ca2+ influx [+ T cells of healthy donors, stimulation by anti-CD3/anti-CD28, caused a transient increase of the TRPM2 expression [2+ overload and triggering the intrinsic pathway of apoptosis, so TRPM2 is considered as a tumor suppressing factor [TRPM2 form nonrectifying channels, almost equally permeable to Kand Ca2+  and sligCa2+ [2+ . TRPM2 iCa2+ [2+ . (TRPM2)eukemia) , 228. TReukemia) . The com+ influx . In CD4+pression . TRPM2 mpression . Overexpg factor .+ and K+ [2+ for its activation [+ cells; TRPM5 was expressed in stimulated and nonstimulated PBLs and Jurkat. TRPM4 and TRPM5 possess similar biophysical characteristics and are unique among TRP superfamily, which are virtually Ca2+ impermeable [+ influx, producing membrane depolarization [2+ influx. Indeed, suppression of TRPM4 in Jurkat transforms PHA-induced Ca2+ signal from oscillations to a higher sustained Ca2+ increase, resulting in a higher IL-2 production [2+, but, in contrast to the former, it is strongly suppressed by external acidification [TRPM4 encodes an intrinsically voltage-dependent outwardly-rectifying  monovale+ and K+ . It requtivation . Wenningermeable . Thus, urization . This teoduction . Similarfication , which mfication . Interesfication . In addification . However In vivo current, generated by TRPM7 channels, is strongly outwardly rectifying, due to a blockage by cytosolic Mg2+ and, possibly, some additional internal factor [2+ block of TRPM7 channel and a role of a very similar TRPM6 channel in Mg2+ homeostasis led to a hypothesis that TRPM7 could equally participate in sensing and control of the intracellular Mg2+. Yet, current experimental evidence is more against that in favor of this hypothesis [2+ or Ca2+ influx through its pore, kinase domain activity, or a combination of these factors. Interestingly, TRPM8, a crucial motor element for cell migration in glioblastomas [TRPM7 is another, in addition to TRPM2, “chanzyme,” consisting of ionotropic divalent cation-permeable channel and kinase, linked to cytosolic C-terminus . It is ul factor . Mg2+ blpothesis , 213. TRpothesis . Importapothesis . Althouglastomas  but oppolastomas  for a relastomas .2+-selective TRPV5/6 ones. TRPV1 (vanilloid receptor), the founding member, represents an outward-rectifying voltage-gated channel, which is upregulated by different physical factors  and a great variety of unrelated chemical compounds  Ca2+ levels [v1.3, KCa3.1, and CRAC, thus contributing to a variety of vital T cells functions by modulation of the Ca2+ signaling [2+-dependent process, unlike mature peripheral lymphocytes, either mouse or human, where hypotonic stress does not provoke any intracellular Ca2+ change [2+ signal it is known that it is mediated by the plasma membrane nonselective channels (TRPs?), which are 100-time less sensitive to Gd3+ as compared to the CRAC. Likewise, in immature T cells and T-ALL the volume regulation is controlled by a mechanosensitive TRP, which, in accordance with our data, could be the TRPV2.TRPV1 and TRPV2 are the most expressed among TRPV1/4 channels in human peripheral blood and leukocytes, with the TRPV2 mRNA being more abundant than that of TRPV1 by a factor of 150 and 20, respectively , 244. TR release . There a release . TRPV2 i and Na+ . In huma and Na+ . It is t and Na+ , 251. Ho+ influx , 253. Ac+ influx . Our own+ influx , albeit  tissues –258. It + levels . It is s+ levels . We give+ levels , but thiignaling . In Jurk2+-selective (PCa/PNa > 100) members of the TRP family [TRPV5 and 6 are inwardly rectifying and the only highly CaP family , 215. BoP family . TRPV6 iP family .2+-permeable channels, activated by external ATP [2+ influx, was obtained by Baricordi and coworkers (1996). It was demonstrated that P2X may contribute to T cell proliferation, induced by mitogenic stimulation [+ and Jurkat cells [2+ rise and enhances IL-2 production [2+ influx, NFAT activation, and interleukin production [2+ entry, providing an amplification mechanism for TCR signaling [P2X forms nonselective Carnal ATP . First emulation . There aat cells , 264. T at cells  expressioduction , 264. Sioduction , 264. Thignaling , 264. ATignaling  via someignaling  or, notaignaling . Additioignaling .Remarkably, the evidences emerged, which link P2X7 with leukemogenesis. Bone marrow samples obtained from patients with different types of leukemias, mainly AML and CLL, show a substantially higher level of P2X7 mRNA expression as compared to normal donor group . Quite a+) transport needs to be accompanied by a parallel transport of anions via anion channels. In ALL (T-ALL) the expression of ClC2-5 was detected by PCR; ClC-3 is robustly expressed in healthy PBL and leukemias, whereas ClC-2 is mainly in leukemias [− channels, whereas in case of leukemic cells  it is mediated by DIDS-insensitive NPPB-sensitive ones  [swell)  was first discovered in peripheric human T cells [− current for CFTR (cystic fibrosis transmembrane conductance regulator) cAMP-activated [lck) activity: the inhibition of this kinase blocks swelling-induced Cl− current, whereas addition of p56lck to excised patches caused the current activation [− channel and not a pump. Transfection of the CFTR-defective B cells and lymphoblasts with wild-type CFTR cDNA resulted in a restoration of a cell-cycle-dependent  cAMP—activated outward rectifying Cl− current [− current can be activated in a triple manner: transiently by cAMP and steadily by either hypotonic stress or intracellular Ca2+ increase; only the activation by cAMP was abolished by genetic defect in CFTR [2+ increase in response to T cell receptor activation. This may be a depolarization, caused by outward-rectifying Cl− current which normally moderates Ca2+ influx via CRAC [− channels are expressed in normal lymphocytes and lymphoblasts. Whereas in case of ClC channels direct demonstration of respective currents is lacking, in case of VSOR its molecular identity remains to be revealed. Preliminary pharmacological analysis suggests differential dominant Cl− currents in normal and malignant lymphocytes.Any osmotic or volume adjustment  requires a massive transport of solute across the plasma membrane. Such a massive transport is possible only when it occurs in electroneutral manner, so that cations  , 270. VS T cells –273. Idectivated  and volutivation . Once activation –276; sin current , 278. It in CFTR . In addivia CRAC . Summari+ channels [α-subunits of voltage-dependent L-type Ca2+ channels (Cav1) as well as interacting β-subunits is consistently reported in human healthy T and Jurkat cells. However, up to now neither depolarization-activated Ca2+ selective currents nor significant Ca2+ influx evoked by a depolarization was detected in human T cells [2+ currents, which disappeared upon Cav1.4 silencing, were reported in a single study [β-subunits or Cav1.4-deficient mice, or mice with knock-down Cav1.2 and/or Cav1.3 display clear immunopathological phenotypes , or inducing apoptosis (dexamethasone). Allogeneic bone marrow transplantation has been shown to be beneficial in a selected population in this leukemia subtype. Although the leukemia-free survival has significantly improved in the last decades, being reported in some series in children up to 85% at 5 years , traditi in vitro apoptosis induction capability in LLA cells lines that express high levels of FLT3 and interacting synergistically with multiple chemotherapeutic agents. JAK inhibitors are currently in varying stages of development and they have only been in adult trials. Notch inhibitors are under development. Though, in animal models and in the phase I clinical trials, serious gastrointestinal toxicity has been shown, which can be reversed by concomitant use of glucocorticoids, subsequent additions have been few and mainly restricted to the advent of some new chemotherapeutic drugs , the use of monoclonal antibodies such as alemtuzumab, and more recently drugs able to impact molecular targets . Sustair review , 108. Hor review , 77, it r review .2+ signaling. Channels may be involved in the cell motility and tumor metastasis. These phenomena may provide the basis for the targeting ion channel in leukemogenesis.Leukemias show altered expression of a variety of ion channels , and modification of the channel protein, as well as every aspect of microenvironment and metabolic regulation and signaling pathways context, affecting the channel's activity, may be of potential importance for the tumor progression. Here, the integration of the channels function and their crosstalks, most apparently via cell membrane polarization and intracellular Ca2+ changes, needs to be considered.Tumor-specific expression of a certain ion channel is a relatively rare phenomenon. However, changes in the expression (including its variation during cell cycle) and subcellular localization, splicing of channels (and relative expression of different variants), role of heteroligomerization, of


The aim of the present study was to characterize the effect of CBG on intracellular Ca2+ ([Ca2+]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence on ICa-L, Ca2+ transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10−8 M) decreased the maximal inhibition of CBG by 47.93%. CBG reduced ICa-L in a concentration-dependent manner with an IC50 of 4 × 10−10 M, upshifted the current-voltage curve of ICa-L, and shifted the activation and inactivation curves of ICa-L leftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca2+ transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca2+ channels, and reduced [Ca2+]i and contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca Bufo melanostictus Schneider or Bufo bufo gargarizans Cantor) i) homeostasis. This Ca2+ overload is characterized by a rise in [Ca2+]i to a level that triggers pathological events in the cell, such as arrhythmias, mechanical dysfunction , and eventually cell death i levels were inferred from the ratio of the fluorescence intensity at two wavelengths (340/380). Ca2+ transients were calibrated as described previously i. Abundant Ca2+ is released into cardiomyocytes within a short period of time from inner stores in the SR via Ca2+-induced Ca2+ release. This instantaneous rise in Ca2+ consequently activates the Ca2+ pump in the SR and Na+-Ca2+ exchanger in the cytomembrane. Finally, the Ca2+ pump and Na+-Ca2+ exchanger transport the Ca2+ into the SR and out of cardiomyocytes, and the [Ca2+]i decays immediately thereafter. This phenomenon is termed the Ca2+ transient i (Figures ICa-L, Ca2+ transient, and contraction in rat ventricular myocytes also were all observed within the range of safe concentrations.However, previous studies on CBG generally focused on its cardiotoxicity. CBG and other bufanolide steroid compounds have been reported to have similar chemical structures to digitoxigenin  and to p Figures . This fiICa-L in a concentration-dependent manner and to reduce Ca2+ transients and cell contraction in ventricular myocytes. The results reasonably suggest that the inhibitory effects of CBG may have therapeutic benefits and support a possible link between the cardioprotective effects of CBG and Ca2+ channels. The findings also provide experimental evidence for rational applications of Kyushin and SBP for patients in the clinic. Because the CBG-mediated mechanism of ICa-L suppression was not directly determined in the present study, further work is necessary to better understand the precise mechanism for the inhibitory effects of CBG on ICa-L.In summary, CBG was demonstrated to have significant inhibitory effects on


Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca2+-mediated signalling. Ca2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuBKU80, were engineered to express the bioluminescent Ca2+-reporter aequorin. An aequorin-mediated method for routine Ca2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca2+]c) in response to extracellular stimuli were measured. The response to extracellular challenges  that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca2+]c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca2+-signalling components in each stress response. Concordant with the involvement of Ca2+-calmodulin complexes in these Ca2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca2+-signatures to all the challenges. The Ca2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca2+ in initiating the stress responses. Aspergillus fumigatus causes multiple human and animal diseases, the manifestations of which depend upon a complex interplay between pathogen- and host-mediated activities c) dynamics was previously developed for living cells of filamentous fungi  using synthetic codon-optimized aequorin as a bioluminescent Ca2+-reporter [A. fumigatus cells for routine [Ca2+]c measurement by multiwell plate luminometry during the early stages of colony initiation; (ii) characterise the dynamic intracellular Ca2+-signatures produced by the fungus in response to a range of environmental stimuli that might be experienced by A. fumigatus during lung infection; (iii) determine the influence of extracellular Ca2+ and calmodulin on generating these Ca2+-signatures; and (iv) analyse the effects of these environmental challenges and pharmacological treatments on the growth of the fungus.A routine method for cell population-level measurements of cytosolic free calcium  or Aspergillus Complete Medium (ACM)  was flanked by 800 bp of the 5’ and 3’ regions of the akuBKU80 gene (AFUA_2G02620) to direct homologous recombination to the akuBKU80 locus. The 5’ flank is identical to the genomic DNA sequence occurring at -800 to -1 bp of the region upstream of the akuBKU80 ATG codon, and the 3’ flank is identical to the DNA sequence occurring at +188 to +988 bp of the region downstream of the akuBKU80 stop codon  (hypo-osmotic shock), (3) 5 mM, 20 mM or 200 mM of CaCl2 in AMM , (4) 2.5 mM, 5 mM, 10 mM and 20 mM H2O2 in AMM (oxidative stress), (5) 12.5%, 25% or 50% v/v human serum diluted in AMM (exposure to human serum), or (6) 50 mM, 250 mM or 500 mM mannitol in AMM (hyper-osmotic stress). The Ca2+-chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid) tetrapotassium salt (BAPTA) (5 mM) and the calmodulin antagonist trifluoroperazine (TFP) (50 μM) were obtained from Sigma . Stock solutions were prepared in water as appropriate and BAPTA and TFP were added as a pretreatment for 30 min prior to exposure to each stress condition.Conidia of the 2+]c in populations of conidial germlings during an experiment, the following empirically derived equation from Bonora et al., 2013  and the number of Ca2+ binding sites on aequorin molecule, all of which are assumed to be identical to values previously described c using the above formulae has been included as To convert relative light units (RLUs) into dynamic measurements of micromolar c dynamics in A. fumigatus, we constructed a Ca2+-reporter strain in two different A. fumigatus genetic backgrounds, the wild-type clinical isolate CBS 144–89 (CEA10) and a non-homologous end re-joining mutant ΔakuBKU80, a null mutant in the AFUA_2G02620 locus, which exhibits significantly heightened homologous integration frequencies compared to non-mutated A. fumigatus isolates c transients and thus different Ca2+-signatures  or oxidative stress (5–20 mM) the [Ca2+]c did not fully recover to the pre-stimulatory resting levels within 10 min of the initial application of the stress stimulus (see below). The highest [Ca2+]c amplitude was prompted by treatment with 200 mM extracellular CaCl2 . Similar results were also achieved using the AEQΔakuB strain (data not shown).Each stressor prompted immediate and distinctly different c increase, fungal cells activate Ca2+-pumps and-transporters in order to attempt to restore [Ca2+]c to a resting level of 0.05–0.1 μM [2+]c decreases following the application of each stressor varied between the different types of stimuli applied. After exposure to 500 mM mannitol (hypertonic shock), [Ca2+]c slowly decreased to resting level over ~ 3 min  within the 10 min of measurement -ethane-N,N,N’,N’-tetraacetic acid tetrapotassium salt (BAPTA) and the calmodulin antagonist trifluoperazine (TFP). BAPTA chelates Ca2+ ions in the extracellular medium thereby preventing the fungus from using extracellular Ca2+ during Ca2+-signalling. TFP, upon binding to calmodulin, induces a major conformational change in the Ca2+-calmodulin complex which results in its inability to interact with target enzymes c amplitude following application of the different stressors, and the post-stimulatory [Ca2+]c resting levels at 10 min after stressor application, were analysed in untreated, BAPTA (5 mM) and TFP (50 μM) pre-treated samples , hyper-osmotic shock (500 mM mannitol), oxidative stress (20 mM H2O2) and 50% human serum  after exposure to high (200 mM) extracellular Ca2+ and oxidative stress (20 mM H2O2) suggesting that [Ca2+]c responses are mediated by calmodulin in response to these treatments  in 5 μl of water were inoculated and colonial size and morphology evaluated after 72 h of growth at 25°C , BAPTA treatment decreased hyphal growth rate in liquid culture in the absence of a challenge. Most importantly, consistent with a role for extracellular Ca2+ in mediating the response of A. fumigatus to these stresses, BAPTA treatment significantly perturbed hyphal growth rate in liquid culture when hyphae were challenged with H2O2 and 50% human serum  level of [Ca2+]c measured was 0.05–0.1 μM which undoubtedly does not correspond to the high free Ca2+ concentration present in organelles such as vacuoles, reported to be roughly 2.5 mM, which is 25,000-fold higher than the concentration in the cytosol (46). Moreover, no targeting sequence was added to the aequorin gene and thus we expect the aequorin to be exclusively cytosolic. In this study, an A. fumigatus aequorin-expressing strain was constructed by modification of the clinical isolate CBS 144–89 (CEA10), thereby achieving a targeted and single integration of the aequorin gene into the A. fumigatus genome , the product of which governs the rejoining of non-homologous DNA fragments. The resultant AEQΔakuB strain provides a recipient reporter strain, in which targeted deletion of desired genes can be easily achieved using standard molecular techniques and repair of pyrimidine auxotrophy. This auxotrophic recipient achieves similar growth to prototrophic isolates when supplemented with uridine and uracil .The bioluminescent Capeptides ,43,52–55s genome . In ordeckground . The Δak2+]c dynamics by challenging the cells with an array of different stressors, likely to be encountered by A. fumigatus conidial germlings in the environment and in the mammalian lungs during infection. The Ca2+ signatures associated with each physico-chemical treatment was found to be distinct, suggesting the involvement of a different combination of Ca2+-signalling machinery components in each stress response  result in the recruitment of the transcription factor, CrzA, to nuclei, which up- and down-regulates a range of genes which support stress adaptation  show increased sensitivity to oxidative stress caused by H2O2 or menadione, and decreased expression of oxidative stress response genes, such as GLR1 (glutathione reductase), TRR1 (thioredoxin reductase) and IPF7817 (NAD(P)H oxidoreductase family protein). In Schizosaccharomyces pombe, deletion of cmk2, which encodes a serine-threonine kinase related to Ca2+/calmodulin-dependent kinases, results in hypersensitivity to oxidative stress c, and have demonstrated correlations between external stimuli, specific changes in [Ca2+]c and either the inhibition or stimulation of growth. The mechanistic basis of how specific [Ca2+]c signals are generated, and converted into specific responses, in filamentous fungi remains to be addressed. The recent development of genetically encoded Ca2+-sensitive probes that can be used to image subcellular [Ca2+]c dynamics in filamentous fungi c response. Average values ± SD error for six technical replicates are shown. The arrows indicate the point at which each stress was applied via the injectors of the plate reader. Bar: 10 μm.Each stressor was applied at two time-points of growth (24 and 28 h) at 25°C. Cultures were also microscopically analysed in order to compare the stage of conidial germination and germ tube growth with the [Ca(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 FigA. fumigatus AEQCEA10 cultures were pre-treated for 30 min with either 5 mM BAPTA or 50 μM TFP prior to challenge with stressors (applied at points indicated by arrows). Note the influence of the modulators on the [Ca2+]c amplitudes and post-stimulatory [Ca2+]c resting levels. Statistical comparisons between untreated and treated samples are presented in (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


P86L‐CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca2+ removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L‐CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca2+ signal pathway; (ii) cell survival proteins ERK1/2 and Ca2+/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca2+ concentrations ([Ca2+]n) and cytosolic Ca2+ concentrations ([Ca2+]c) on Ca2+ entry conditions, we observed that baseline [Ca2+]n was higher in CALHM1 and P86L‐CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca2+]c levels in HeLa cells overexpressing CALHM1 and P86L‐CALHM1 compared with control cells. Treatment with Aβ elicited a significant decrease in the cell survival proteins p‐ERK and p‐CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L‐CALHM1‐overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aβ, P86L‐CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro‐cytotoxic pathways, thus potentially contributing to its deleterious effects in AD.The mutated form of the Ca CALHM1 is expressed in all brain regions and neuronal cells, at the ER, and in the plasma membrane. CALHM1 generates Ca2+‐selective cation currents in the plasma membrane. It has also been shown to form a novel Ca2+‐permeable ion channel, whose gating is allosterically regulated by both membrane voltage and extracellular Ca2+ concentration; in addition, CALHM1 is insensitive to classic selective blockers of voltage‐gated Ca2+ channels, although it is inhibited by nonselective and inorganic Ca2+ channel blockers such as Co2+ , ras/mitogen‐activated protein kinase (MAPK), ERK1/2 , and cell survival at baseline or after treatment with Aβ. We used transfected HeLa cells with the empty vector (control) and cells transfected with vectors including CALHM1 and P86L‐CALHM1 to study the kinetics of the changes of [Ca2+]c and [Ca2+]n generated by reintroduction of Ca2+ and treatment with Aβ. We also analyzed ERK, CREB activation, and apoptosis pathways upon exposure to Aβ. Our results indicate that P86L‐CALHM1 could contribute to neuronal vulnerability by affecting cytosolic and nuclear Ca2+ homeostatic mechanisms and survival signaling pathways.As Ca2+ compartments such as cytosol    was 1.47‐fold and 2.27‐fold higher in CALHM1 and P86L‐CALHM1 cells, respectively, than in control cells  and in P86L‐CALHM1 cells (τoff 3.41‐fold higher), with respect to control cells  is a key mechanism of Ca2+ entry into the nucleus. As the ER membrane forms part of the nuclear envelope and CALHM1 is anchored to the ER, we explored whether CALHM1 or P86L‐CALHM1 overexpression could affect the kinetics of the [Ca2+]n transients elicited by indirect InsP3R activation by histamine. We first perfused cells with a Ca2+ solution (1 mm) for 2 min and replaced this solution with another one containing 100 μm of histamine for 15 s. Figure 2+]n variations elicited by histamine‐InsP3R stimulation. Once more, baseline [Ca2+]n was higher in CALHM1 and P86L‐CALHM1 cells than in control  and its subsequent addback (1 mm Ca2+) in the absence or presence of Aβ.Previous results gave rise to the hypothesis that CALHM1 could behave as a leak channel regulating changes in the kinetics of nuclear [Ca2+ elicited a significant increase in the [Ca2+]c, reaching 4.34 and 1.25 μm in CALHM1 and P86L‐CALHM1, respectively  and 1 mm extracellular Ca2+, slight oscillations in baseline [Ca2+]c in both CALHM1 and P86L‐CALHM1 cells were detected (data not shown). Extracellular Ca2+ was then withdrawn and this protocol repeated in the presence of Aβ; Ca2+ entry was significantly reduced in CALHM1‐ or P86L‐CALHM1‐overexpressing cells  to 1.75 μm (plus Aβ), whereas in P86L‐CALHM1 cells, the peak was reduced by 44% from 1.25 to 0.55 μm. In control cells, [Ca2+]c changes were mild and similar in the presence or absence of Aβ  .In the absence of Aβ, the addback of Ca1–42, 5 μm for 24 h, only P86L‐CALHM1‐overexpressing cells showed significant cell toxicity (24% of cell death) compared with cells overexpressing the empty vector or the wild‐type channel , CALHM1, or P86L‐CALHM1 Fig. A. When bet al., We also evaluated vulnerability to oxidative stress stimuli using phenylarsine oxide (PAO), which causes oxidative stress via a mitochondria‐dependent mechanism (Vay 1–42 (5 μm) for 24 h. We observed a clear tendency toward early triggering of apoptosis only in cells overexpressing the mutated form P86L‐CALHM1 at 3 and 6 h (data not shown). Thus, we incubated the cells overexpressing CALHM1 and P86L‐CALHM1 for a longer time period to determine how long it would the apoptosis stage upon treatment with Aβ. After 24 h, only P86L‐CALHM1‐overexpressing cells activated the early apoptosis pathway  for 24 h. .To clarify the mechanism involved in the cell death observed in Fig 1–42  μm for 21–42 5 μm for 8 h, the activity of caspases 3 and 7 rose significantly only in cells overexpressing P86L‐CALHM1 versus CALHM1 . However, treatment with Aβ significantly decreased both p‐ERK expression and t‐ERK expression in P86L‐CALHM1 cells  133 by several kinases, including ERK. In P86L‐CALHM1 cells treated with Aβ, we observed significantly lower expression of p‐CREB than P86L‐CALHM1 cells not treated with Aβ  were somewhat higher in the cells expressing both forms of CALHM1; the differences detected are similar to those observed with [Ca2+]c under the same experimental conditions . It therefore seems plausible that CALHM1 behaves as a leak Ca2+ channel and that the greater baseline [Ca2+]n levels in CALHM1 and P86L‐CALHM1 cells could be explained by Ca2+ leakage from the ER lumen into the nucleus. This interpretation is in line with the idea that the nucleus might have specific Ca2+ transients at specific functionally distinct subcompartments; in fact, the nuclear reticulum can generate localized nuclear Ca2+ gradients . Wild‐type coelenterazine was purchased from Biotium . 4′6‐diamidino‐2‐phenylindole (DAPI), Aβ25–35, anti‐β‐actin, histamine, human Aβ1–42, paraformaldehyde, propidium iodide (PI), thiazolyl blue tetrazolium bromide (MTT), TritonX‐100, and Tween 20 were purchased from Sigma . The antibodies anti‐c‐Myc and anti‐CALHM1 and the PVDF membranes were from Millipore , and Alexa 488 and Image iT‐FX signal enhancer were from Thermo Fisher Scientific . The BCA Protein Assay Kit Reagent was from GE™ Healthcare, and the ECL Advance™ Western Blotting Detection Kit was from Fisher Scientific. The DAKO® mounting medium was purchased from DAKO . The antibodies anti‐CREB, anti‐P‐CREB, and anti‐P‐ERK were from Cell Signalling ; anti‐total ERK and secondary antibodies were purchased from Santa Cruz Biotechnology . We analyzed apoptosis using the FITC Apoptosis Detection Kit . Other general chemicals were purchased from Sigma  or Panreac Química S.L.U. . The cDNA encoding for aequorins was a gift from Professor Tullio Pozzan (University of Padua). The cDNA encoding for CALHM1 and P86L‐CALHM1 was a gift from Professor Philippe Marambaud .Metafectenem glutamine, 25 U mL−1 penicillin, and 25 μg mL−1 streptomycin .HeLa cells were grown in plastic flasks in DMEM supplemented with 10% fetal bovine serum, 2 m4 cells seeded on 12‐mm‐diameter coverslips and grown to 60–70% confluence. Transfection with the genetically encoded photoprotein aequorins targeting the nucleus (nu_AEQ) or cytosol (cyt_AEQ) was achieved using Metafectene® as described elsewhere for cells  ; the zero Ca2+ solution contained 0.5 mm ethylene glycol tetraacetic acid. To induce entry of Ca2+, KHB deprived of Ca2+ was switched to another solution containing 1 mm CaCl2, as specified in the figure legends. When used, 10 μm Aβ25–35 or 100 μm histamine was added to the KHB. Light emission was measured in a purpose‐built luminometer and calibrated in terms of [Ca2+], as described by  (5 μm) for 1 h and then harvested and lysed to determine the expression of CREB, pSer133CREB, ERK1/2, and pERK1/2 (Western blot).HeLa cells were seeded on 24‐well plates and transfected as described above; 24 h after transfection, cells were treated with a mixture of protofibrils and oligomers of Aβ1–42 at 5 μm was achieved as previously described  at 2.3 mm; an aliquot of the 2.3‐mm solution was then dissolved in PBS to a final concentration of 80 μm. Finally, this solution was incubated at 37 °C for 2 h under constant shaking.Aggregation of Aβet al., 4 HeLa cells were seeded in 48‐well plates and transfected with 0.5 μg of empty vector, CALHM1, or P86L‐CALHM1. Twenty‐four hours after transfection, cells were treated with 5 μm of aggregated Aβ1–42, oligomycin (10 μm) plus rotenone (30 μm), or phenylarsine oxide  for 24 h. Cells were then incubated with MTT reagent for reducing for 30 min to form formazan crystal, which was then dissolved with DMSO. Optical density (OD) was read using an ELISA reader at 540 nm . Cell viability was expressed as a percentage of the control and calculated using the following equation: viability = [OD test/OD baseline] × 100.Cell viability was measured using an MTT assay as described elsewhere , which is based on the ability of annexin V to bind specifically to phosphatidylserine flipped into the outer layer of the plasma membrane and the ability of the nonvital dye propidium iodide (PI) to bind to DNA only in altered membrane cells. Thus, double staining enables discrimination between intact cells (annexin V–negative and PI‐negative), early apoptotic cells , and late apoptotic cells (annexin V‐positive and PI‐positive) or necrotic cells (without the characteristic cell integrity).5) cells were seeded in 6‐well plates and transfected with 0.75 μg of empty vector and 2 μg of CALHM1 or P86L‐CALHM1. Twenty‐four hours after transfection, HeLa cells were treated for 24 h with 5 μm of a mixture of protofibrils and oligomers of Aβ1–42 and collected and incubated with fluorescent annexin V and PI. Apoptosis was determined by flow cytometry.In brief, HeLa . Activation of caspases 3 and 7 is expressed as a percentage of nontreated transfected cells.We measured the activation of caspases 3 and 7 using a commercial kit based on luminescence (Caspase‐Glo (R) 3/7 Assay, Promega Biotech Ibérica S.L., Madrid, Spain) as described by  containing albumin 4% and incubated with anti‐p‐ERK, anti‐total ERK, anti‐P‐CREB, or anti‐total CREB and anti‐β actin for 2 h. After washing several times with TTBS, the corresponding secondary antibodies were added for 45 min. Finally, the membranes were revealed using the Western Blotting Detection Kit (Thermo Fisher Science) and analyzed and quantified using Scion‐Image software .HeLa cells transfected with 0.5 μg of empty vector, CALHM1, or P86L‐CALHM1 and treated or not with 5 μt‐test or anova and Bonferroni's, Dunnett's, or Tukey's tests in a post hoc analysis. Differences between experimental groups were considered statistically significant at P < 0.05.Values are given as mean ± SEM. The statistical differences between means were assessed using the This work was partly supported by the following grants: Ministerio de Economía y Competitividad, FPU Program, Refs. AP2009/0343 (AJMO) and AP2010/1219 (IB). ARN: FIS PI10/01426. MGL: Ministerio de Economía y Competitividad, Ref. SAF2012‐23332. MFCA: Consolidación de grupos de investigación UAM‐CAM 1004040047. We also thank Fundación Teófilo Hernando, Madrid, Spain, for their continued support.None declared.Fig. S1 Cellular localization of CALHM1 and P86L‐CALHM1.Click here for additional data file.Fig. S2 Cell vulnerability after treatment with Aβ25–35 in HT‐22.Click here for additional data file.Fig. S3 Early apoptosis triggered by treatment with Aβ25–35 in CALHM1‐ and P86L‐CALHM1–expressing HT‐22 cells.Click here for additional data file.Fig. S4 Dose response curve of Okadaic Acid.Click here for additional data file.Data S1 Supplemental Material and Methods.Click here for additional data file.


Mutations in mtDNA, aberrant expression and/or regulation of Ca2+-handling/transport proteins and abnormal Ca2+-dependent relationships among the cytosol, endoplasmic reticulum, and mitochondria can cause the deregulation of mitochondrial Ca2+-dependent pathways that are related to these processes, thus determining oncogenic behavior. In this review, we propose that mitochondrial Ca2+ remodeling plays a pivotal role in shaping the oncogenic signaling cascade, which is a required step for cancer formation and maintenance. We will describe recent studies that highlight the importance of mitochondria in inducing pivotal “cancer hallmarks” and discuss possible tools to manipulate mitochondrial Ca2+ to modulate cancer survival.Cancer is sustained by defects in the mechanisms underlying cell proliferation, mitochondrial metabolism, and cell death. Mitochondrial Ca Ca2+ reer (MCU) , 10.2+ signaling was significantly remodeled, compromising mitochondrial functions in ways that caused them to overwhelm normal cells. Uncontrolled growth, programed cell death evasion, metabolic reprograming, invasion, and metastasis are “cancer hallmarks” that are linked to mitochondrial Ca2+ remodeling, which could be the prime reason for these transformations  are juxtaposed to mitochondrial Ca2+ handling/transport , toward the matrix-specific Ca2+ transporter, where these channels participate in structures devoted functionally to facilitating Ca2+-flow toward the mitochondrion  and by topological organization, mitochondria are able to accumulate Cah the ER , where transport . Voltagehondrion . This fl complex . MCU is  complex , 10, and complex . MCU/MCU+-uptake –20. Fina+-uptake .2+ requires efficient mechanisms to turn off the signal to prevent the mitochondrial permeability transition pore (mPTP) opening. The most well-characterized Ca2+-efflux mechanism involves the mitochondrial Na+/Ca2+ exchanger   . Ongoingsynthase . The maneostasis .2+ ions are central to both events  and oxidative phosphorylation. An increase in mitochondrial ATP levels was shown to occur in parallel with an evoked increase in mitochondrial [Ca2+] . The firoduction , 28. Theproposed .2+ of TCA: Pyruvate dehydrogenase (PDH), isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. All of these molecules exhibit different Ca2+-dependent mechanisms, suggesting that the origins or specific activation signals are quite dissimilar. Although the deregulation of mitochondrial metabolism is intimately linked to oncogenic behavior, the role of cellular metabolism in oncogene-induced transformation is unclear. Recently, the mitochondrial gatekeeper PDH was shown to be a crucial mediator of oncogene-induced senescence promoted by BRAF (V600E), which is the oncogene that is commonly mutated in tumors  counteracting the effects of Bcl-2 on ER Ca2+ filling . The upregulation of HK expression in tumor cells and its binding to VDAC provide both metabolic benefits and apoptosis-suppressive capacity, offering advantages in terms of growth and resistance to chemotherapy  with no deleterious effects. Oxidative stress is considered an important factor for additional “apoptotic signal” (2+ flux (Much remains to be understood regarding the additional signals that converge on mitochondria and switch their function to apoptotic inducers. These organelles can handle large Ca signal” , 63. ROS signal” , 65. Ind(2+ flux .2+.A growing number of tumor suppressor genes and oncogenes have been investigated due to their ability to regulate mitochondrial function, influencing the physiological processes of this organelle and its capacity to uptake Ca2+ regulation and oncogenic Ras has been shown by Rimessi et al., in which H-Ras affects the correct transmission of Ca2+ waves from the ER to mitochondria (Among these genes, increasing evidence has highlighted the role of the oncogene Ras in the growth and maintenance of the tumor environment . The firchondria . As a rechondria  and by echondria –75.2+-dependent apoptotic response (2+ release and apoptosis, avoiding mitochondrial Ca2+ overload (2+ release via IP3R3. In this study, PTEN was shown to directly interact with IP3R, counteracting the reduced IP3R-dependent Ca2+ release mediated by AKT phosphorylation in a phosphatase-dependent manner (2+ flux (Additionally, the oncogene AKT regulates cell growth and apoptosis, and upon PI3K stimulation, it rapidly accumulates in mitochondria , where iresponse . In addioverload , prefereoverload . AKT is overload . PTEN wat manner . Anothert manner , promote(2+ flux . An othe(2+ flux .2+-dependent pathway. Different reports have documented the ability of p53 to regulate cell fate via post-translational modifications (2+ transfer from ER to mitochondria. An extranuclear pool of p53 interacts with the Ca2+-ATPase SERCA, modulating Ca2+-dependent ER–mitochondria crosstalk, mitochondrial swelling and apoptosis induction (Additionally, the tumor suppressor p53 can regulate tumorigenesis in a Caications , 88; hownduction , 90.in vivo and in vitro, enhances apoptosis susceptibility by acting directly on the mitochondrial compartment. Specifically, FHIT increases the affinity of the mitochondrial machinery for physiological agonist- and apoptotic challenge-triggered Ca2+-uptake into mitochondria (The tumor suppressor FHIT functions in a different manner. This hydrolase, which acts as a tumor suppressor chondria .2+ ion regulates various cellular processes, some of which are involved in tumorigenesis and which are thus considered as attractive drug targets for cancer therapy. Mitochondrial Ca2+ homeostasis may be affected by anti-cancer drugs that directly influence mitochondria or targets that indirectly regulate mitochondrial Ca2+-uptake. Some of these drugs may affect mitochondrial Ca2+ uptake due to the depolarization of ΔΨ. The compounds included in this category are as follows: pancratistatin (a natural alkaloid exhibiting anti-cancer effects against human colorectal adenocarcinoma xenografts (O-octadecyl-2-O-methyl-racglycero-3-phosphocholine; its redistribution to the mitochondria in HeLa cells causes mitochondrial depolarization and apoptosis (The Canografts  and colonografts , 94], a nografts ], PMT7 [nografts ], edelfopoptosis ], and mapoptosis .2+-uptake indirectly through the induction of ER stress. For example, tunicamycin induces the accumulation of unfolded proteins in the ER, which is known to enhance antitumor effects of different chemotherapy drugs in vitro and in vivo (2+-stores (2+ homeostasis, or affects IP3R activity. Bortezomib, a selective proteasome inhibitor approved for use in patients with multiple myeloma, requires the mitochondrial uniporter as critical regulatory factor for its cytotoxicity (Anti-cancer drugs can also influence the mitochondrial Ca in vivo . The can in vivo , or by t+-stores . Brefeld+-stores , causes toxicity .2+ effects of Bcl-2 are attributable to direct interactions with IP3R through the BH4 domain (2+-dependent apoptotic response in a variety of tumoral cells (The anti-apoptotic Ca4 domain , 104. A al cells –108.The HK–VDAC complex is also an oncological target of erastin, which is an antitumor agent that is selective for tumor cells bearing oncogenic Ras , and of 2+-uptake were obtained by potentiating MCU complex expression/activity. Unfortunately, recent molecular discoveries have impeded the identification of an MCU-specific inducer. Alternative approaches could include MCU-targeting anti-miRNA-based therapy and anti-miRNA 25 oligonucleotides that could be used as potential agents against cancer (The most direct effects of potential anti-cancer drugs on mitochondrial Cat cancer .2+ remodeling, which could be the prime reason for these transformations.” This speculation in the Section “in vivo (2+ internalization is ΔΨ, which is generated by respiratory chain, mitochondrial Ca2+ signaling has become a prime factor that is responsible for oncogenic behavior. Although mitochondrial Ca2+ remains a novel area of research in oncology, considerable further work is required to clarify the molecular mechanisms by which it contributes to cancer formation and maintenance, and greater knowledge of this target may be of importance in the development of new therapeutic strategies.“Uncontrolled growth, programed cell death evasion, metabolic reprograming, invasion, and metastasis are ‘cancer hallmarks’ that are linked to mitochondrial Ca“in vivo , thus hiThe 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.


Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1–7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1–7) agonist, AVE0991, in experimental cirrhosis.4) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991.Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride  in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways . Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats.The non-peptidic agonist of Ang-(1–7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure. The renin-angiotensin system (RAS) plays a pivotal role in cirrhosis and portal hypertension. Increased hepatic and systemic levels of angiotensin (Ang) II activates profibrotic and contractile pathways downstream of the AngII type 1 receptor (AT1R) on hepatic stellate cells (HSCs), promoting fibrosis and contraction. This, in turn, results in an increased vascular resistance .7-Ang-(1–7) . –11. 9–11–7, a metng-(1–7) ,12,13. Fng-(1–7) .in vitro that AVE0991 reduced hepatic stellate cell (HSC) activation and collagen production [AVE0991 is a non-peptidic specific Ang-(1–7) agonist which can be given orally and has a much more prolonged half-life than the heptapeptide. In the kidney and lung, AVE0991 induces the release of nitric oxide (NO) and acts as an anti-inflammatory and antifibrotic agent via MasR stimulation –19. Studin vivo with regard to portal hypertension in two different models of cirrhosis in rats.Thus, we evaluated a potential therapeutic efficacy of long-acting MasR stimulation in cirrhosis models ad libitum  with free access to water.We used 89 Sprague-Dawley wild type (WT) rats. Experimental procedures were approved by the Animal Ethics Committee of Austin Health and of North Rhine-Westphalia (LANUV 84–02.04.2011.A164). WT rats were housed in a controlled environment  and fed standard rat chow 4 for 14–16 weeks until ascites was present as described previously [4.Eight rats (100g) underwent twice weekly inhalation of 1l/min CCleviously ,21. Ten BDL was performed in 40 rats (180g) after anesthesia with ketamine/xylazine (78mg/kg and 10mg/kg body weight) as previously described . BDL ratWhen rats developed ascites as a definite sign for the presence of portal hypertension, rats were used for the hemodynamic studies as described previously . To asseBriefly rats were anesthetized with ketamine/xylazine (78mg/kg and 10mg/kg body weight), respectively. To investigate hemodynamics, the coloured microsphere technique was performed as described previously [4 intoxicated WT rats. Additionally we collected pieces of AVE0991 treated rat livers directly after performing the microsphere technique. Western blot analysis  and reverse transcribed to cDNA by use of a protocol previously described  and in STM) [Referring to Maul et al.  the actiTM) ,26. The Osmotic mini-pumps  were filled with AVE0991  dissolved in polyethylene glycol 400  and sterile water. The pumps were filled with 200μl of AVE0991 solution and incubated in saline overnight at 37°C were implanted in 14 rats in the peritoneal cavity during BDL surgery and delivered 28μg/kg/h for 2 weeks as previously described [In analog segments (200mg) of snap-frozen livers, the hepatic hydroxyproline content was determined colorimetrically as described previously  and in SFor the detection of collagen fibers, paraffin-embedded sections of liver (4μm) were stained with picrosirius-red and the proportion of Sirius-red staining per field at was assessed at ×100 magnification in a total of ten fields per section. This was performed in a blinded fashion using computerized image capture , as described previously .Bile ductular proliferation was scored using the grading system  detailedp-values < 0.05 were considered statistically significant.Data are presented as mean±standard error of the mean (s.e.m.). Student’s t-test was used for comparison where appropriate; Mann-Whitney-U test was used for comparison between groups. in vivo hemodynamics we used the BDL (4–5 weeks) and the CCl4 intoxication (14–16 weeks) models when the presence of ascites indicated the presence of severe portal hypertension. Cirrhotic rats  showed the typical signs of cirrhosis and portal hypertension with an increased portal pressure compared to controls  underlining the mechanism for the hepatic vasodilation in cirrhosis  and its activity, measured by the phosphorylation of its substrate Moesin, were significantly reduced after acute injection of AVE0991 in both models of cirrhotic rats, as well as in sham-operated control rats .4 intoxicated rats  showed higher bile ductular proliferation score compared to sham-operated rats, however administration of AVE0991 via osmotic mini-pumps for 2 weeks had no effect on the biliary proliferation score in fibrotic BDL rats Fig B in .in vitro and in vivo in renal, cardiovascular and endothelial research [in vivo.This study showed for the first time, that acute administration of the non-peptidic Ang-(1–7) agonist AVE0991 reduced portal pressure in cirrhotic rats without causing systemic side effects. This effect was associated with an up-regulation of the p-eNOS/NO pathway and an attenuation of the Rho-kinase pathway within the liver. The effects of AVE0991 are well characterized research ,18,30–33in vivo and liver perfusion pressure ex vivo in cirrhotic livers [in vivo, leading to a significant reduction of portal pressure. Either a strong association of AT1R and MasR or crosstalks of their downstream pathways might explain these data. Further studies are required to highlight this interaction in detail.We have recently reported that Ang-1–7) lowers portal pressure in cirrhotic rats –7 lowersc livers . This wac livers . It has c livers . Indeed,c livers . Therefoc livers . These min vivo. This increase in splanchnic vascular resistance might be a direct AVE0991 effect (possibly via interaction with AT1R), or it might be a splanchnic vascular response to the marked decrease of intrahepatic resistance.Besides the upregulation of MasR in the cirrhotic liver, increased MasR expression was described in the splanchnic-vascular bed of cirrhotic animals and humans . Indeed,Furthermore, the systemic and peripheral circulation was not affected by AVE0991. This was surprising since AVE0991 has been described to be a potent vasodilator in different pathological extrahepatic conditions  ,19,30–33These differences between the effects of Ang-(1–7) and AVE0991 might be explained by a number of factors. In comparison to Ang-(1–7), the imidazol derivate AVE0991 has a longer half-life due to its resistance to proteolytic enzymes. The differences between AVE0991 and Ang-(1–7) are also illustrated by the fact that AVE0991 does not directly antagonize vasoconstrictive AngII effects in the liver , while tin vitro work using HSCs that showed a blocking effect of AVE0991 on αSMA and collagen transcription [in vivo. One possible speculative explanation might be, that AVE0991 blunts the expression and activity of ACE/MasR axis as shown in the acute experiments. However, in other models, AVE0991 might have an effect on hepatic fibrosis.In contrast to our previous In summary, we could show that AVE0991 is a potential therapeutic agent to lower portal pressure without extrahepatic systemic effects. This nonpeptidic compound should be further investigated in patients with portal hypertension.S1 Figs(DOCX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 Tables(DOCX)Click here for additional data file.


Previously we reported that acute exposure to BPA promotes arrhythmogenesis in female hearts through alteration of myocyte CaObjective: We sought to define the cellular mechanism underlying the nonmonotonic dose response of BPA in the heart.Methods: We examined rapid effects of BPA in female rat ventricular myocytes using video-edge detection, confocal and conventional fluorescence imaging, and patch clamp.2+ transient, were characterized by nonmonotonic dose responses. Interestingly, the effects of BPA on individual processes of myocyte Ca2+ handling were monotonic. Over the concentration range of 10–12 to 10–6 M, BPA progressively increased sarcoplasmic reticulum (SR) Ca2+ release and Ca2+ reuptake and inhibited the L-type Ca2+ current (ICaL). These effects on myocyte Ca2+ handling were mediated by estrogen receptor (ER) β signaling. The nonmonotonic dose responses of BPA can be accounted for by the combined effects of progressively increased SR Ca2+ reuptake/release and decreased Ca2+ influx through ICaL.Results: The rapid effects of BPA in cardiac myocytes, as measured by multiple end points, including development of arrhythmic activities, myocyte mechanics, and Ca2+ handling processes. This represents a distinct mechanism underlying the nonmonotonicity of BPA’s actions.Conclusion: The rapid effects of BPA on female rat cardiac myocytes are characterized by nonmonotonic dose responses as measured by multiple end points. The nonmonotonic dose response was produced by ERβ-mediated monotonic effects on multiple cellular Cahttp://dx.doi.org/10.1289/ehp.1307491Citation: Liang Q, Gao X, Chen Y, Hong K, Wang HS. 2014. Cellular mechanism of the nonmonotonic dose response of bisphenol A in rat cardiac myocytes. Environ Health Perspect 122:601–608;  Bisphenol A  is one of the highest production volume chemicals worldwide, with annual production exceeding 3 million metric tons. BPA is used in the production of polycarbonate plastics, epoxy resins, and nonpolymer additives to other plastics. It is used extensively in the manufacturing of common consumer products and goods such as food containers, metal cans (as a protective coating), beverage and baby bottles, receipt paper, and water pipes . Human e2+ handling, including elevated sarcoplasmic reticulum (SR) Ca2+ spontaneous release (or Ca2+ leak) and SR Ca2+ reuptake, are a key mechanism underlying the proarrhythmic action of BPA  with potentially adverse impacts on human health . A largen of BPA . These fPreviously we reported that the rapid effect of BPA on the contractility of female cardiac myocytes is characterized by a nonmonotonic dose response . A nonmoReagents. All reagents and solvents used were of the highest purity available. Aqueous solutions were prepared using BPA-free water . Bisphenol A (BPA) obtained from TCI America  was provided by the Division of the National Toxicology Program (DNTP) at the National Institute of Environmental Health Sciences. Methyl-piperidino-pyrazole,1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) and 4-pyrimidin-3-yl]phenol (PHTPP) were obtained from Tocris Cookson , and nifedipine was purchased from Sigma-Aldrich . Isopreterenol, nifedipine, and other chemicals were from Sigma-Aldrich unless otherwise stated.Animals. Animal procedures were performed as previously described by ad libitum with Teklad 2020 diet , which contains no soybean meal, alfalfa, or animal products that may introduce uncontrolled levels of estrogenic compounds. Sterile drinking water was generated by a dedicated water purification system (Millipore Rios 16 with ELIX UV/Progard 2) that reduces oxidizable organics to < 1% of source levels. Drinking water was dispensed from glass water bottles. A total of 70 animals were used in this study.2+ handlingAnalysis of myocyte mechanics and Ca. Ventricular myocytes from female rat hearts were enzymatically dissociated using Langendorff perfusion, as previously described  and imaged with a Zeiss LSM 710 inverted confocal microscope  with an excitation wavelength of 488 nM. Signals were measured with line-scan imaging at 3.07-msec intervals, with each line comprising 512 pixels spaced at 0.056 mm. Image processing and data analysis were performed as previously described . Experiments were performed at room temperature (24°C).escribed , 2013. Iescribed . Brieflyescribed . To measCaL (L-type Ca2+ current)Patch clamp recording of I. The ICaL was recorded at room temperature (24°C) using the whole-cell patch clamp technique, as previously described .escribed . After tWestern blotting. Western blotting experiments were performed as previously described  with differences between treatment groups assessed using a multiple comparison posttest. Frequency of events  was analyzed using a chi-square test. We considered p < 0.05 the minimal level of statistical significance for differences in values. Data were analyzed using SigmaPlot  and Excel .Rapid actions of BPA in cardiac myocytes have nonmonotonic dose responses. We examined the concentration-dependent effect of BPA on Ca2+ transient in female rat ventricular myocytes. BPA rapidly (~ 5 min) increased the amplitude of the Ca2+ transient at low BPA doses (10–10 to 10–8 M), and this stimulatory effect diminished at micromolar doses  of 0.81 nM and a maximum increase of 209.7% , PLN is the central regulator of SR Ca2+ reuptake. PLN can be phosphorylated by both protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CAMKII) at serine 16 and threonine 17, respectively. Phosphorylation of PLN releases its inhibition on SERCA, thereby increasing Ca2+ reuptake into the SR   . The aveBPA’s rapid actions are mediated by ERβ signaling. Previously we hypothesized that the opposing actions of estrogen receptor (ER) α and ERβ contribute to the nonmonotonic dose response of BPA in the heart  on ICaL  of nifedipine of 0.3 μM  had significant impact on arrhythmogenesis and the mechanics of female cardiac myocytes, and that the dose responses of the rapid impact of BPA are nonmonotonic. The nonmonotonicity is produced by multiple monotonic effects on individual cellular Ca2+ handling processes through ERβ-mediated signaling  declines compared with 10–8 M; 10–6 M BPA was not statistically different (p > 0.2) compared with control  plus nifedipine to mimic the effect of micromolar BPA, and that the overlapping effects of BPA and nifedipine on ICaL do not fully reproduce the effect of micromolar BPA. Nevertheless, the result qualitatively demonstrates that the nonmonotonic effect of BPA can be accounted in part by ICaL inhibition.Growing evidence has provided increasing understanding of the mechanisms that generate the nonmonotonic dose response of EDCs. The known mechanisms are diverse and have been reviewed in a number of articles . Example process . Previout of BPA . The preizations . The exachanisms . To testCaL by high-dose BPA is similar to that observed in response to high concentrations of 17β-estradiol (E2), suggesting the possibility of a common mechanism. Several studies have shown that supraphysiological E2 (10–30 μM) partially suppresses ICaL in cardiac myocytes in multiple speci, including rat, human, and guinea pig (2 has been shown to suppress ICaL in guinea pig ventricular myocytes, with an IC50 of 29.5 nM and a maximum suppression of 40% (CaL by high doses of estrogens, while of limited physiological relevance, may play a role in determining the dose–response properties of other estrogenic chemicals in the heart.Suppression of Iinea pig . E2 has n of 40% , values 2+ handling processes is sufficient to account for the observed nonmonotonicity, the potential contribution of other mechanisms, such as receptor desensitization, were not examined in the present study and cannot be ruled out. In addition, the molecular/signaling mechanisms underlying BPA’s impact on the Ca2+ handling elements are unknown and remain to be elucidated.It has been shown both experimentally and computationally that nonmonotonic dose responses can be generated by the opposing actions of multiple receptors . In prev2+ handling processes, all mediated by ERβ signaling. The summation of these parallel effects generated an inverted U-shaped dose response, with the most efficacious dose around 10–8 to 10–9 M, coinciding with reported human exposure levels (The rapid effects of BPA on female rat cardiac myocytes were characterized by nonmonotonic dose responses, as measured by multiple end points; the cellular mechanism of BPA’s nonmonotonicity involves monotonic and opposing effects on multiple Cae levels  (Figure e levels .


In the last decade, considerable advances have been made in the investigation of dipolar quantum gases. Previous theoretical investigations of a rotating binary dipolar Bose-Einstein condensate, where only one component possesses dipole moment, were mainly focused on two special orientations of the dipoles: perpendicular or parallel to the plane of motion. Here we study the ground-state and rotational properties of such a system for an arbitrary orientation of the dipoles. We demonstrate the ground-state vortex structures depend strongly on the relative strength between dipolar and contact interactions and the rotation frequency, as well as on the orientation of the dipoles. In the absence of rotation, the tunable dipolar interaction can be used to induce the squeezing or expansion of the cloud, and to derive the phase transition between phase coexistence and separation. Under finite rotation, the system is found to exhibit exotic ground-state vortex configurations, such as kernel-shell, vortex necklace, and compensating stripe vortex structures. We also check the validity of the Feynman relation, and find no significant deviations from it. The obtained results open up alternate ways for the quantum control of dipolar quantum gases. In a cold dilute atomic gas, atoms predominantly interact with each other through the short-rang van der Waals interaction, which can be described in terms of elastic EC)152Cr μB, 168Er), 168Er μB, and 1nd 164Dy μB with μd-wave collapse and expansion13161719202122s-wave scattering length between −∞ and +∞, leading to the realization of a purely dipolar gas. Furthermore, it is experimental realization that the DDI can be reduced its natural value, including to negative values, by external field rotation, which leads to a large parameter spaceDue to the long-range and anisotropic nature of the DDI, ultracold atoms with a magnetic (or electric) dipole moment exhibit a variety of intriguing phenomena, such as the anisotropic deformation and excitation11One of the characteristic properties of superfluid is that they respond to rotation by forming quantized vortices, which is a universal feature of macroscopic quantum systems with spontaneously broken U(1) symmetry. Vortex physics becomes much more diverse in dipolar gases than in typical short-range interacting ones, due to its long-range and anisotropic2526272831So far, most of the previous studies (including the aforementioned works) on dipolar BEC have been restricted to single- or multi-component system, where each component has a dipole moment. Very recently, the ground-state and rotational properties of a binary dipolar gas, wherein only one component possesses magnetic dipole moment, have drawn considerable attentions35363738non-dipolar condensate.Motivated by such rich physics and to fill up this gap, it is of particular interest to extend the situation to an arbitrary orientation of the dipoles with respect to the plane of motion, which is what we attempt to do in this work. Our results show that the ground-state phases and vortex structures of such a system depend strongly on the relative strength between the dipolar and contact interactions, and the rotation frequency, as well as on the orientation of the dipoles. The inclusion of DDI introduces another “switch”, which can be used not only to obtain the desired ground-state phase, but also to control the number of vortices and its related vortex structures, even for a ψ1 and ψ2. We consider a binary BEC confined in a quasi-two-dimensional (Q2D) plane, with the polarization axis being at angle Θ to the x axis in the x-z plane. The Q2D regime is obtained by assuming a sufficiently strong trapping potential in the axial direction z, thus the z dependence of ψ1 and ψ2 can be factored out and all the relevant dynamics occur only in x and y directions. Hence we arrive at the effective Q2D nonlocal GP equations for the order parameters of a binary dipolar BEC4041Our studies are restricted to the zero-temperature mean-field regime, where the ground-state and dynamics of a binary dipolar BEC are described in terms of two complex-value order-parameter ψi is the condensate wave function, mi is the atomic mass, Ωi is the effective rotation frequency of component i, and Lz = −iħ(x∂y − y∂x) is the z component of the orbital angular momentum operator. The Q2D external potential can be written as mR = m1m2/(m1 + m2) being the oscillator length in the z direction and the reduced mass, describe the intra- and inter-component interactions, respectively. To simplify the numerical analysis and highlight the effects of DDI, it is convenient to reduce the number of free parameters in the problem. Thus, we specialize to the “balanced” case where ωx1 = ωx2 = ωy1 = ωy2 = ω, m1 = m2 = 2mR ≡ m, N1 = N2 = N, Ω1 = Ω2 = Ω, and consider only the fixed repulsive short-rang contact interactions, i.e., g11 = g22 = g21 = g12 = g = 5543effective contact interactions between atoms can be controlled by modifying atomic collisions , or the atom number N. In addition, we assume that component 2 is nondipolar, leading to μ0 and μ are the magnetic permeability of vacuum and the magnetic dipole moment, respectively. For dipolar component 1, we assume all the dipoles are aligned to some external field, which is in the x-z plane, and forms an angle Θ with the x-axiswhere εdd = μ0μ2m/12πħ2as, which determines the physical properties of such a component. Due to the unprecedented level of experimental control of the strengths of DDI and contact interactions, in principle, such a parameter can be tuned from positive to negative values5εdd < 1, the short-range part of the interparticle interaction dominates and DDI provides only corrections, which corresponds to a stable BEC. However, large values of εdd may make the system unstable against collapse for small values of the angle Θ, due to the anisotropic nature of the DDI.For the dipolar component 1, the most important quantity (dimensionless) is the relative strength between the dipolar and contact interactions, Now we consider the ground-state and rotational properties of such a system as a function of tunable DDI and its orientation, and of the rotation frequency, and then propose how to obtained the desirable ground-state by synchronizing the DDI and rotation frequency in future experiments.switch, is found to give rise to novel density distributions.Let us first investigate the ground-state structure of the system as a function of the dipolar strength, and of the dipoles of the atoms with respect to the plane of motion, for fixed contact interactions. To highlight the effects DDI, the system is first set as rest without rotation. It is well-known that for the nonrotational case, the ground-state density distributions of the system depend sensitively on the relative strength between inter- and intra-component contact interactions, showing phase coexistence or phase separation. However, the presence of the tunable dipolar interactions in component 1, worked as another effective εdd = 0.3, 0.55, 1.0, 1.45. First, it is clear from x-axis (Θ = 0°), we observe the well-known self-induced squeezing of the cloud along the y-axis direction, as shown in the last row of 26x-axis but repulsive in the y-axis, leads to an effectively anisotropic effect. On the other hand, the presence of repulsive inter-component interaction between the dipolar and scalar components, together with the anisotropic DDI, result in the squeezing of the dipolar component and the spatial phase separation of such two components.x- and y-axis. When the orientation of the dipoles exceeds Θ = 75°, the behavior of the system changes, the repulsive part of the dipolar interaction becomes dominant, leading to a net effective repulsion. As expected, this effect is maximal when the dipoles are polarized along the z axis with Θ = 90°. In this special case, the dipolar interaction is purely repulsive and isotropic, leads to an isotropic expansion of the cloud.Upon increasing Θ slowly to 30° and 45°, one find a similar, but less pronounced, change of the density profile, as in these cases the attractive part of the dipolar interaction becomes less important. Further increases of Θ, such as Θ = 60°, the situation is changed. In this case, the repulsive and the attractive parts of the dipole interaction become comparable, leading to a nearly isotropic expansion of the cloud along both the x2〉/〈y2〉 to describe the self-induced squeezing. y-axis direction performs more obviously for dipolar component, while the picture is reversed for non-dipolar one. In addition, when the dipoles are polarized along the z axis, we always get an isotropic cloud, regardless of its value.To give a more clear explanation of the above results, we introduce a dimensionless parameter 〈g12 significantly affects the atom density distributions, even for a trapped binary dipolar condensate.Hence we conclude that when the system is set as rest without rotation, not only the strength of DDI but also its orientation can be regarded as two “degrees of freedom”, which can be used to induce the squeezing and isotropic (anisotropic) expansion of the cloud, and to derive the phase transition between phase coexistence and phase separation. Moreover, the inter-component parameter It is also of interest to investigate the rotational properties of the system as a function of the rotation frequency, which manifest itself as the presence of quantized vortices. Thus we now turn our attention to the effect of rotation frequency on the ground-state vortex configuration of such a system. It is found that the vortex lattice will form with a structure strongly dependent on both the tunable DDI and the rotation frequency.εdd within the range that we have considered. Take Ω = 0.3 as an example, in this case the system shows phase separation at a relatively small value εdd = 1. However, our results below show that further increasing the rotation frequency Ω will induce new nucleated vortices in both dipolar and scalar components.For a relatively slow rotation, we find a similar behavior for the density distribution of such two components as the nonrotational case. The only difference is that the phase separation occurs at a relatively small i.e., Θ = 90°. εdd, showing three typical vortex structures. From these images, we observe that in this case the system is always in phase separation. Interestingly, the density distribution of such a system exhibits a “kernel-shell” structure, in which the dipolar component occupy the outside regime and forms a shell-like structure, while the scalar component occupy the center regime with a structure strongly dependent on the strength of the DDI. It should be mentioned that the “kernel-shell” structure has been previously observed in a rotating two-component dipolar BEC, where dipolar component mainly occupy the center regimeg′ leads to a variation of inter-component interaction, whereas in the present study the inter-component interaction g12 is fixed. In addition, we assuming the dipoles are aligned along some arbitrary and tunable direction, whereas only vertical and parallel situation were considered in the latter case.At first, we consider the situation where the dipolar atoms in the first component 1 are polarized perpendicular to the plane of motion, εdd = 0.3 get increased to four for εdd = 0.55, 1.0, where different vortex structures are formed to lower the total energy of the system.Further information can be extracted from the phase distribution and the density difference of such two components [see the lower panel for each plot of εdd = 1.0. For Θ = 30°, again we find the self-induced squeezing of dipolar component. However, compared with the nonrotational case, three vortex lines  forms due to the presence of rotation. As the orientation of the dipoles increases, stripe pattern appears, as shown in Next we study the effects of the orientation of the dipoles on the rotational properties of the system. In our numerical simulations, it is found that for fixed rotation frequency and the strength of the dipolar interaction, the ground-state vortex structure of the system, as a function of the orientation of the dipoles, shows similar behavior. εdd = 0.9. At Θ = 0°, no regular lattice structure forms for the dipolar component; whereas the vortices (including hidden one) in the scalar component form three vortex lines, where two bend lines present even parity with respect to the central one , as shown in Finally, we illustrate the ground-state vortex structure of the system for a rapid rotation Ω = 0.9. In this case, a rapid rotation will generate a dense array of vortices, which are arranged spontaneously as a vortex lattice to minimize the total energy of the systemAs the orientation of the dipoles increases, the ground-state profiles change gradually into an alternatively arranged stripe pattern, where both the dipolar and scalar atoms are arranged into compensating parallel stripes. It is easy to find that the numbers of vortices in both the dipolar and scalar components increases with increasing Θ. Meanwhile, the locations of the vortices of one component are at the region of the density domains of the other component, and the vortices of each component are overlapped in lines and the density forms a stripe pattern, as shown in 33g can be controlled by modifying atomic collisions, or the atom number N. For the dipolar component, the most important quantity is the relative strength between the dipolar and contact interactions εdd. Thus the parameter sets used in our manuscript illustrate well the possible ground states. Furthermore, it is also of interest to investigate the validity of the Feynman relation for the mean vortex density. For a two-component system, it naturally generalizes toLast but not least, we want to note that while the numerical results presented above are limited to some special values of the system parameters, qualitatively similar behavior is also observed over a range of different parameter sets. Actually, as discussed above, for the non-dipolar condensate, the effective contact interactions between atoms m1 and m2 are the masses of the bosons in the two constituent condensates, and Ω1 and Ω2 are the angular rates at which the two superfluids are rotating19224849m1 = m2 = m, and Ω1 = Ω2 = Ω, which leads to the same areal density of vortices for such two components, as shown in where In light of these results we can better understand the combined effect of rotation and DDI on the ground-state vortex arrangement, and conclude that use of a binary dipolar BEC, wherein only one component possesses dipole moment, is another possible way of creating novel vortex pattern formation without suffering a dipolar collapse52x-z plane, forming an arbitrary and tunable angle with x-axis.In summary, we have investigated the effects induced by the anisotropic nature of the dipolar interaction in a rotating binary dipolar BEC, which consists of both dipolar and scalar bosonic atoms and confined by a quasi-two-dimensional harmonic trapping potential. We have considered the dipole moment to be oriented on the switches, which can be used to quantum control of the ground-state density of such a system. Upon the increases of the rotation frequency, the new nucleated vortices enter each component, and the system exhibits exotic ground-state structure, such as kernel-shell, vortex necklace, and compensating stripe vortex structure, due to the anisotropic characteristic of the dipolar interaction. Finally, we have checked the validity of the Feynman relation of the system and found no significant deviations from it.We have analyzed the ground-state and rotational properties of the system for different strength of DDI and its orientation, and for different rotation frequency. For the nonrotational case, our results show that the strength of DDI and the orientation of the dipole moment, worked as two different mJ = −J and mJ = 0. With regard to the relative strength between the DDI and contact interaction, typically, for 52Cr with magnetic dipole moment d = 6μB and s-wave scattering length as ≈ 100a0 (a0 being the Bohr radius), εdd = 0.036One point of particular importance is that our results may shed insight on problems focusing on the interplay between isotropic inter-component and anisotropic dipolar interactions, which has not been studied as thoroughly. To estimate the physical realizability of the described effects, perhaps the most promising system is a binary BEC, which can be realized by selecting two states in the ground hyperfine manifolds of atomic Cr, Dy, or Er, where components 1 and 2 consist of states with spin projections The long-range and nonlocal DDI for component 1 has the form θ is the angel between the polarization direction and the interatom vector r − r′. To deal with this DDI, it is convenient to work in Fourier space and use the convolution theorem3955where F(q) with k space DDI for the Q2D geometry, which consists two parts coming from polarization perpendicular or parallel to the direction of the dipole tilt. That is α being the angle between qd is the wave vector along the direction of the projection of x-y plane, and erfc is the complementary error functionwhere −7. In the numerically simulations, we work in dimensionless units by introducing the scales characterizing the trapping potential: the length and time are scaled as To study the ground-state and rotational properties of the system, we start from proper initial wave-functions and use the split-step method within an imaginary-time propagation approach5960How to cite this article: Zhang, X.-F. et al. Exotic vortex lattices in a rotating binary dipolar Bose-Einstein condensate. Sci. Rep.6, 19380; doi: 10.1038/srep19380 (2016).