Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disorder characterized by prolonged fever, cytopenia, hepatosplenomegaly, rash, icterus, and other neurological symptoms. Successful treatment of HLH by etoposide has improved outcomes for children with HLH. However, the development of treatment-related acute myeloid leukemia (t-AML) after the usage of this drug is a concern.We report a case of acquired EBV-triggered HLH with progression to t-AML following etoposide therapy with cytogenetic abnormality for t   resulting in MLL gene fusion. The development of t-AML was detected 23 months after HLH diagnosis.Although the successful treatment of HLH by etoposide has improved outcomes for children with HLH, t-AML is a serious complication of topoisomerase II inhibitor therapy and is characterized by the presence of gene rearrangement. This study suggests that HLH patients undergoing therapy with HLH-2004 protocol need monitoring for future malignancy, especially in the initial 2 to 3 years. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disease of the immune system. HLH can be related into inherited gene defects or be triggered by severe infections, particularly with Epstein-Barr virus (EBV), rheumatoid disorders, or various malignant neoplasms. Familial hemophagocytic lymphohistiocytosis (FHL) is an immune disorder of autosomal recessive inheritance and is usually diagnosed within the first two years of life. Disease-causing mutations in gene PRF1, UNC13D, STX11, RAB27A, and STXBP2 (Munc18-2) have been found in FHL [The successful treatment of HLH by etoposide has improved outcomes in children with HLH. However, development of t-AML related to the usage of this drug is a concern. In the present study, a case of EBV-HLH is reported with progression to t-AML following etoposide therapy with cytogenetic abnormality for t   resulting in MLL gene fusion.9/L. A magnetic resonance imaging (MRI) of the brain revealed no abnormality. Polymerase chain reaction (PCR) analysis for the EBV EBNA-1 was positive in peripheral blood. The EBV-DNA in blood was 6.25 × 104 IU/mL. Bone marrow examination showed increased hemophagocytic histiocytes (HS)  at 12,108 U/L, total bilirubin at 100.9 umol/L, direct bilirubin at 51.5 umol/L, fibrinogen at 390 mg/dl, PT/PTT at 20.5/65.4 s, thrombin time of 46.7 s, and D-dimers at 851 ng/L. In summary, the patient fulfilled 5 out of 8 HLH-2004 criteria [HS) Fig. , which i2 during the 16-week period.The patient received chemotherapy according to the HLH-2004 protocol, which includes etoposide, steroids, intrathecal methotrexate, and cyclosporin. The patient achieved complete remission of the disease at the end of the initial treatment period of 16 weeks. The patient did not achieved full remission of the disease until the end of the initial treatment period of 8 weeks. Treatment was thus continued until week 16 and was then stopped asl the parents declined further therapy. In total, the patient’s cumulative dose of etoposide was 2100 mg/mAfter 23 months, the patient was readmitted with fever, leukocytosis, anemia, diffuse lymphadenopathy, and hepato-splenomegaly of 5 and 4 cm, which is palpable below the costal margins. LDH was elevated at 1788 U/L. Bone marrow examination revealed 89 % monoblasts, without evidence of HS Fig. . Flow cyThe patient is currently on chemotherapy for AML, and her bone marrow revealed no residual or recurrent leukemia cell after the initial complete remission.We report a case of a 10-month-old Chinese female with EBV-associated HLH, who showed a progression to t-AML following etoposide therapy with cytogenetic abnormality for t   and MLL gene fusion, 23 months after the onset of HLH.The t-AML following the etoposide therapy is not infrequent in malignant neoplasms such as leukemia and has first been reported in lung cancer in the 1980s . About 2The aim of this paper is to fully describe the patient with an adverse effect of etoposide in the therapy of HLH and to characterize the risks and prognosis of t-AML.2. Imashuku et al. [2 (P = .0001), compared with that of <1000 mg/m2 and without etoposide.HLH is a life-threatening hyperinflammatory disease caused by an uncontrolled and dysfunctional immune response of inherited or acquired immune deficiencies. All forms of HLH are characterized by an impaired function of natural killer (NK) cells and cytotoxic T-cells (CTL) . EBV-assu et al.  reportedHowever, development of t-AML related to the usage of this drug is a concern. Most translocations of leukemia associated with epipodophyllotoxin disrupt an 8.3 kb breakpoint cluster region (b c r) between exons 5 and 11 of the MLL gene at chromosome band 11q23 . MoleculAdditional chromosome aberrations, such as t 15; 17), t , inv (16), t , t , or t , were also reported in younger patients and showed a shorter latency period between the primary tumor and t-AML . Four of5; 17, t Cases of t-AML following epipodophyllotoxin treatment generally occur within a relatively short latency period (ranging from 6 to 36 months), with 11 out of 13 cases developing t-AML within 36 months, as shown in these findings  showed that the calculated cumulative 6-year risks for the occurrence of t-AML for the low (<1.5 g/m2), moderate (1.5–2.99 g/m2), and high dose (≥3.0 g/m2) groups were 3.2, 0.7, and 2.2 %, respectively, and concluded that the incidence of t-AML after treatment with epipodophyllotoxins is not purely dose-dependent [Different factors play a role for the risk of etoposide-related AML, which include the cumulative dose, schedule, other chemotherapeutic agents, and host factors. Imashuku et al.  investigependent . Winick ependent  investigependent . Geneticependent , 25.The outcome of t-AML is generally considered to be poorer than that of de novo AML. Remission is 80–90 % in t-AML, but long-term survival rates were 10–20 % . ChildreIn conclusion, therapy-related AML is the most serious complication of VP-16/etoposide-related chemotherapy for HLH.CTEP, the national cancer therapy evalution program; CTL, cytotoxic t-cells; EBNA-1, Epstein-Barr virus nuclear antigen-1; HLH, hemophagocytic lymphohistiocytosis; HS, hemophagocytic histiocytes; HSCT, hematopoietic stem cell transplantation; MLL, mixed-lineage leukemia; MTX, methotrexate; NK, natural killer cells; OS, overall survival; PCR, polymerase chain reaction; T-AML, treatment-related acute myeloid leukemia


The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9  significantly upregulated mTOR pathway activity. This was in line with increased accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) and secretion of VEGF and TNF-α. Similar results were obtained in primary human healthy leukocytes. Importantly, in both types of primary cells, Tim-3-mediated effects were compared with those induced by lipopolysaccharide (LPS) and stem cell factor (SCF). Tim-3 induced comparatively moderate responses in both AML cells and healthy leukocytes. However, Tim-3, like LPS, mediated the release of both TNF-α and VEGF, while SCF induced mostly VEGF secretion and did not upregulate TNF-α release. The immune receptor T-cell immunoglobulin and mucin domain 3 (Tim-3) is involved in a variety of leukocyte biological responses . In T ceInterestingly, Tim-3 is highly expressed in leukaemia cells, especially in T-cell lymphocytic leukaemia cells as well as acute myeloid leukaemia (AML) cells –6. In leTim-3 is considered as one of the possible AML antigens, since malignant AML cells preserve the ability to express the SCF receptor (Kit or CD117) as well as Tim-3 , 7. In cex vivo systems such as primary human AML cells versus healthy human leukocytes have not yet been elucidated.In myeloid cell lines and primary myeloid hematopoietic cells Tim-3 is known to mediate the activation of NF-kB transcription factor and TNF-α secretion . HoweverIn the present study, we therefore analysed both the total and cell surface expressions of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes obtained from peripheral blood (buffy coats). We found that, in primary AML cells, Tim-3 expression is much higher compared to primary healthy leukocytes. We also observed that Tim-3 receptor molecules were mostly expressed on the surface of primary AML cells, while the majority of Tim-3 protein remained inside primary human healthy leukocytes. In primary human AML blasts (AML-PB001F), Tim-3 agonistic antibody as well as galectin-9  induced activation of the mTOR pathway (by mTOR-dependent phosphorylation of p70 S6 kinase 1 (p70 S6K1) and eIF4E-binding protein-1 (eIF4E-BP1)). This was in line with HIF-1 activation and increased secretion of VEGF and TNF-α. Similar results were obtained in primary human leukocytes isolated from buffy coats obtained from the blood of healthy donors. Importantly, in both types of primary cells, the effects were compared with those induced by lipopolysaccharide  ligand) and SCF (Kit ligand). In primary AML cells SCF induced the strongest biological response, whereas LPS displayed comparatively greater effects on primary human leukocytes. Tim-3 in both cases induced moderate cellular responses. However, although Tim-3, like LPS, triggered the release of both TNF-α and VEGF, SCF induced mostly VEGF secretion and did not significantly impact the TNF-α release.via phosphorylation of its S2448 residue and HIF-1 signalling in human AML cell lines [Our recent work demonstrated that Tim-3 mediates the activation of mTOR In order to compare the expression and, more importantly, re-distribution of Tim-3 in the cells we analysed its total amount and cell surface presence using in-cell Western and in-cell assay respectively. We found that both primary AML blasts and healthy whole blood leukocytes expressed Tim-3 as detected by in-cell Western and in-cell assay Figure . HoweverGiven the high expression/externalisation levels of Tim-3 protein in primary human AML cells compared to PLs we sought to obtain evidence on differences in receptor downstream activities. We found that, in primary AML cells, mTOR S2448 phosphorylation was substantially increased upon exposure to SCF as well as to anti-Tim-3 antibody stimulation (about 50% of SCF-induced effect), while LPS displayed a weaker ability to upregulate the intracellular levels of phospho-S2448 mTOR. The same pattern was observed for PI-3K activity Figure . Activitvia activation of all three pathways, the intensities of which were comparable with mTOR-dependent processes following exposure to SCF  was the most powerful cell activator Figure . This isInterestingly, background intracellular levels of phospho-S2448 mTOR, HIF-1α and Tim-3 proteins as well as VEGF, but not PI-3K activity and secreted TNF-α, were significantly higher in AML blasts compared to healthy PLs  and eIF4E-BP1 (S65), accumulation of HIF-1α protein and secretion of TNF-α and VEGF. It is however, clear that the effects are moderate and are weaker than those induced by LPS or SCF, a classic inflammatory mediator and hematopoietic growth factor, respectively.In AML cells, SCF displayed the highest biological activity while LPS effects were stronger in healthy whole blood PLs. These differences are due to differential expression of TLR4 and Kit receptors in healthy and malignant leukocytes. SCF-induced responses in healthy PLs are likely to involve both direct and indirect effects. Several types of leukocytes express Kit receptors. This includes hematopoietic stem cells, multipotent progenitors, common myeloid progenitors, common lymphoid progenitors, megakaryocytes, myeloblasts, small lymphocytes, eosinophils, NK cells and dendritic cells , 12, 13.Although Tim-3 ligands are known to trigger programmed death of T cells and demonstrate downregulatory properties  and pro-angiogenic (VEGF release) activities . Maxisorp™ microtitre plates were obtained either from Nunc  or kindly provided by Oxley Hughes Ltd . Mouse monoclonal antibodies to HIF-1α, mTOR and β-actin as well as rabbit polyclonal antibodies against phospho-S2448 mTOR were purchased from Abcam . Antibodies against phospho-T389 p70 S6 kinase 1 (p70 S6K1), total and phospho-S65 eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) antibodies were obtained from Cell Signaling Technology . Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from Li-Cor . ELISA-based assay kits for the detection of VEGF, TNF-α and IL-6 were purchased from Bio-Techne . All other chemicals were of the highest grade of purity and available commercially unless otherwise stated.RPMI-1640 medium, foetal calf serum and supplements, Primary human AML mononuclear blasts  were purchased from AllCells  and handled in accordance with manufacturer's instructions. Cells from two different patients were analysed.Primary human leukocytes were obtained from buffy coat blood . The buffy coat blood was purchased from the National Health Blood and Transfusion Service  following ethical approval (REC reference: 12/WM/0319). Mononuclear-rich leukocytes were obtained by Ficoll-density centrifugation according to the manufacturer's protocol. Cell numbers were determined using a haemocytometer and diluted accordingly with HEPES-buffered Tyrode's solution before treatment as indicated below.E.Coli and purified in accordance with published protocols [Human SCF protein was produced in rotocols .Expressions of HIF-1α, total and phospho-T389 p70 S6K1, as well as total and phospho-S65 eIF4E-BP1 were determined by Western blot analysis and compared to β-actin in order to determine equal protein loading, as previously described . Li-Cor 2O2).Phosphorylation of mTOR was analysed by ELISA as recently described . BrieflyWe employed a standard Li-Cor in-cell Western assay to analyse total Tim-3 expressions in primary human AML cells and healthy primary leukocytes (PLs). The in-cell assay was also applied to detect Tim-3 protein surface presence in the studied cells .Concentrations of these cytokines released into the medium were analysed by ELISA (R&D Systems assay kits) according to the manufacturer's instructions.The activity of PI-3K was measured using a non-radioactive assay based on the ability of the enzyme to phosphorylate its substrate as previously described .t test. Statistical probabilities (p) were expressed as *, where p < 0.05, **, where p < 0.01 and *** when p < 0.001.Each experiment was performed at least three times and statistical analysis was conducted using a two-tailed Student's


At parallel-fiber (PF)-to-Purkinje neuron (PN) synapses in the cerebellar cortex loss of calretinin (CR), the major buffer at PF terminals, results in increased presynaptic Ca2+ transients and an almost doubling of the initial vesicular releases probability (pr). Surprisingly, however, it has been reported that loss of CR from PF synapses does not alter paired-pulse facilitation (PPF), while it affects presynaptic Ca2+ signals as well as pr. Here, we addressed this puzzling observation by analyzing the frequency- and Ca2+-dependence of PPF at unitary PF-to-PN synapses of wild-type (WT) and CR-deficient (CR−/−) mice using paired recordings and computer simulations. Our analysis revealed that PPF in CR−/− is indeed smaller than in the WT, to a degree, however, that indicates that rapid vesicle replenishment and recruitment of additional release sites dominate the synaptic efficacy of the second response. These Ca2+-driven processes operate more effectively in the absence of CR, thereby, explaining the preservation of robust PPF in the mutants.Endogenous Ca Contrary to the previous report  was significantly reduced in the mutants, suggesting that a rapid recovery of the releasable vesicle pool (RP) may compensate for its depletion during the first stimulus. Experimentally constrained computer simulations combined with an analysis of successes and failures, and multiple probability fluctuation analysis (MPFA) suggest the involvement of a Ca2+-driven mechanism in PPF  is an endogenous CaB closely related to CB. Four of its binding sites bind Ca−/− mice . Slices were kept for 30 min. at 35°C prior to the experiments. Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich, Seelze, Germany.Horizontal cerebellar slices (300 μm thick) were prepared  from the vermis region of 21–24-day-old C57BL/6 and CRa-receptor blocker gabazine (SR-95531). Experiments were carried out at room temperature. Cells were visualized with an upright microscope  equipped with a 60×/0.9 NA water immersion objective (Olympus). Somatic whole-cell patch-clamp recordings from PNs were obtained using an EPC10-2 amplifier  and “Patchmaster 2.6” software. The liquid junction potential (15 mV) was corrected for. During the experiments, the series resistance and leak current were monitored continuously and experiments were rejected if either the series resistance exceeded 30 MΩ, deviated by more than 15% from its initial value, or the leak current fell below −350 pA.Patch pipettes were prepared from borosilicate glass  with a PC-10 puller  and had final resistances of 4–5 MΩ or 10–11 MΩ when filled with intracellular solution for recordings from PNs or GCs, respectively. The intracellular solution contained (in mM): 150 K-gluconate, 10 NaCl, 3 Mg-ATP, 0.3 GTP, 10 HEPES and 50 μM EGTA dissolved in purified water .The pH was adjusted to 7.3 with KOH. Slices were transferred to the bath chamber perfused continuously at 3 ml/min. with ACSF containing 10 μM of the GABAStimulation of individual GCs was achieved in the loose-cell attached configuration in the voltage-clamp mode by using the current response of the amplifier to positive voltage steps Perkins,  and boroAll experiments were carried out in accordance with institutional guidelines for animal experiments, and were approved by the state directorate of Saxony, Germany.EPSCs were analyzed with “Patchmaster 2.6” software and custom written routines in Igor Pro 6.21 . paired-pulse ratios (PPRs) were calculated by dividing the average of the second EPSC amplitude of a given ISI by the average of the first EPSC amplitude of all ISIs at a given synapse. Amplitudes were determined by fitting a product of two exponential functions to the baseline-subtracted currents, which allowed for independent adjustment of the time constants of the rising and falling phases. The amplitude of the second EPSC was determined as the difference between its peak and the decay of the first EPSC, thereby eliminating the effects of electrical summation in particular at short ISIs. EPSCs were classified as failures if their amplitude was < 5 pA i.e., 2-fold root mean square (RMS) noise. It cannot be excluded that some EPSCs with smaller amplitude remained undetected.2+]e  and a 5 s interval between paired stimulations. In indicated experiments 5 mM of the competitive low-affinity AMPA receptor antagonist γ-D-glutamylglycine (γ-DGG) was added to the bath to relieve postsynaptic receptor saturation.Quantal synaptic parameters were determined from EPSC amplitudes recorded at an ISI of 10 ms at different , using pr of 0.25 for WT PF synapses N]. Thus, rapid replenishment of N  remains unaltered between first and second pulse, the EPSC2/EPSC1 PPR is given by . PPR showed a frequency dependence almost identical to the WT, dropping with a time constant of 100 ms to unity. Consistent with a reduced PPF (and an increased pr) F1 was significantly reduced in CR−/− compared to the WT .To probe whether Ca Sakaba,  or by re Sakaba, ) could epr of 0.41  is ~1.4 in the absence of replenishment. With full replenishment (N2 = N1 => PPR = pr2 /pr) PPRmax would be ~2.4, i.e., the measured PPR substantially exceeds the theoretical PPRmax without replenishment and at an ISI of 5 ms is close to PPRmax with 100% replenishment. F2 in the mutants was significantly reduced compared to WT  and, as in the WT, reached the F1 value at ISI ~300 ms. Yet, it did not drop to 0 even at an ISI of 5 ms (0.09 ± 0.04), i.e., also in the mutants pr2 did not reach a value of one but rather is ≤ ~0.91. This suggests that even a full replenishment of N between pulses is not sufficient to account for the measured PPF in CR−/−. In addition, these data suggest that the control of the size of N is dependent on Ca2+, since it operated more effectively in the absence of CR.PPF in the mutants was still unexpectedly large. Using the above calculations and a 2+-dependent increase in N contributes to PPF, we measured PPF in paired recordings at WT and CR−/− unitary connections at different extracellular Ca2+ concentrations ([Ca2+]e) of 1, 2, and 10 mM at an ISI of 10 ms . PPF was strongest at 1 mM [Ca2+]e with an average PPR of 6.47 ± 1.36 in the WT and 3.56 ± 0.44 in CR−/−. The PPR dropped with increasing [Ca2+]e being only 1.00 ± 0.11 and 0.73 ± 0.08 in WT and CR−/− in 10 mM [Ca2+]e, respectively.To further test the hypothesis that a Cas Figure . Alterat2+]e  which relieves receptor saturation  CR from the simulations without changing other parameters.In our simulations the release-triggering Ca Figures . The maj−/− compared to the WT was obtained  and for CR−/− an increase to 3.5 ± 1 , we performed MPFA of second EPSC amplitudes recorded at an ISI of 10 ms at i) from the second AP, thereby, causing amplified release. However, it has been recognized early that [Ca2+]res alone cannot account for facilitation  and nano- or microdomain [Ca2+]i at the release site during the second AP (tens to hundred μM), simple Ca2+ summation is unlikely to be the major source of facilitation  has been estimated to be ~20 μM at PF synapses, decaying from peak to [Ca2+]res of ~0.5 μM within ~2 ms (Schmidt et al., 2+ and EPSC amplitude, the linear summation of [Ca2+]res and [Ca2+]local can only account for ≤10% facilitation. In addition, it has been estimated that [Ca2+]res decays with an average τ of 42 ms under unperturbed conditions (Brenowitz and Regehr, τ of 100 ms lasted much longer than the elevation in [Ca2+]res (cf. Atluri and Regehr, The size of the release triggering Ca2+]i during repeated synaptic activations (Neher, 2+]i would be expected if Ca2+ current facilitation is involved in the generation of PPF (Bollmann and Sakmann, 2+ sums linearly during repeated activations, both in WT and in CR−/−, making saturation of CR or Ca2+ influx facilitation unlikely to be involved in facilitation of release from PF terminals (Brenowitz and Regehr, The signature of saturation of endogenous CaBs is a non-linear increase in [Cas Neher, . Similar2+ ions (Lou et al., 2+ binding and unbinding, conditioning pulses facilitate these release sensors in the absence of separate facilitation sensors. Thus, the facilitation sensor could be a variant of the facilitated release sensor and not necessarily distinct from the latter one. Considering this, our simulations explain PPF at PF synapses by a synthesis of the two previous suggestions, with the increase in N making the dominating contribution.Specifically for facilitation of release from PF terminals a facilitation sensor (Atluri and Regehr, 2+-driven increase in N may change during postnatal synapse maturation since the active zone and the Ca2+ influx-release coupling undergoes substantial postnatal rearrangement at PF terminals (Baur et al., The magnitude of facilitation caused either by facilitation of the release machinery of non-released vesicles or by Ca2+-binding proteins being considered important regulators of PPF. Consistent with one previous report (Schiffmann et al., pr (Schmidt et al., STP is a central factor in neuronal information processing (Abbott and Regehr, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, it is still not known if atrial and sinoatrial node cells are also involved. In this article we will first briefly review TRPC3 and IP3R signaling that relate to store/receptor-operated Ca2+ entry (SOCE/ROCE) mechanisms and cardiac arrhythmias. We will then present some of our recent research progress in this field. Our experiments results suggest that pacing-induced AF in angiotensin II (Ang II) treated mice are significantly reduced in mice lacking the TRPC3 gene (TRPC3−/− mice) compared to wild type controls. We also show that pacemaker cells express TRPC3 and several other molecular components related to SOCE/ROCE signaling, including STIM1 and IP3R. Activation of G-protein coupled receptors (GPCRs) signaling that is able to modulate SOCE/ROCE and Ang II induced Ca2+ homeostasis changes in sinoatrial complex being linked to TRPC3. The results provide new evidence that TRPC3 may play a role in sinoatrial and atrial arrhythmias that are caused by GPCRs activation.Atrial fibrillation (AF) is a significant contributor to cardiovascular morbidity and mortality. The currently available treatments are limited and AF continues to be a major clinical challenge. Clinical studies have shown that AF is frequently associated with dysfunction in the sino-atrial node (SAN). The association between AF and SAN dysfunction is probably related to the communication between the SAN and the surrounding atrial cells that form the SAN-atrial pacemaker complex and/or pathological processes that affect both the SAN and atrial simultaneously. Recent evidence suggests that Ca In the normal heart, pacemaker cells in the sinoatrial node (SAN) generate spontaneous membrane depolarizations that trigger action potentials, which then propagate through the conduction system to initiate atrial and ventricular cell depolarization and contraction. In contrast to normal pacemaker activity, abnormal arrhythmogenic electrical activity can arise in ectopic sites due to impulse re-entry or abnormal spontaneous membrane depolarizations Nattel, .2+ handling being a key factor  current which not only contributes to pacemaker current(s) but may also produce arrhythmogenic electrical activity , the major cardiac SR Ca2+ release channel, will produce depolarizing NCX current and contribute spontaneous membrane depolarization(s) to feed the genesis of AF   and the phospholipase C (PLC)-IP3 signaling pathway   channel (see below)  gene from 2+ influx can also be triggered as a direct consequence of PLC activation and production of diacylglycerol (DAG) that is independent of SR Ca2+ store depletion and forms a “receptor-operated Ca2+ entry” (ROCE) pathway  liberated from the plasma membrane, triggered by agonist binding to G protein—coupled receptors, such as angiotensin II and endothelin-1 receptors. Treatment with antisense RNA, to inhibit translation of endogenous TRPC3 mRNA, reduces Ca2+ influx activated either by receptor stimulation or passive store depletion   placed on the atria and ventricle  130 NaCl, 1.8 CaClIntact SANs were micro-dissected from right atria as described previously , using established methods  for 15 min to deplete the SR Ca2+ store. SOCE then occurs upon reintroduction of Ca2+ to the perfusate   and TRPC3  in single isolated pacemaker cells. A mouse antibody to STIM1 was also used . Anti-mouse or anti-rabbit secondary antibodies (Alexa-488 anti-mouse and Alexa-594 anti-rabbit (Molecular Probes) were used as secondary antibodies. Prolong gold antifade reagent with DAPI (Molecular Probes) was used as the mounting media and to provide nuclear staining.A rabbit polyclonal antibody was used to label type II IP−/− mice. Ang II was delivered at the rate of 2 μg/g/day for 10–14 days.Alzet osmotic mini-pumps were implanted subcutaneously in WT, or TRPC3n. Statistical tests were either Student's paired or unpaired t-tests, and P < 0.05 was used as the limit of statistical confidence.Data are expressed as mean ± SEM, with the number of preparations as −/− KO mice which had been treated with Ang II (See Methods) over of 10–14 days. Figure −/− respectively, as shown in the selected recordings illustrated in Figure −/−, respectively   Figure .−/− mice supports the idea of a possible involvement of TRPC3 in arrhythmogenesis. As described above, Ca2+ entry through TRPC3 channel activated via a SOCE/ROCE signaling pathway and STIM1 could contribute arrhythmogenic current. To investigate this possibility, we examined expression and molecular localisation of STIM1, and the possibility of molecular interaction between TRPC3, STIM1, and IP3R2 in isolated cardiac pacemaker cells.We previously reported that that pacemaker cells express TRPC3 and it is preferentially localized to the surface membrane  which can modulate pacemaker activity. To examine if IP3R and STIM1 form a signaling complex in SAN we double labeled isolated pacemaker cells with anti-IP3R2 and anti-STIM1 antibodies. Figure 3R2 was similarly distributed and there was some co-localization between these labels (yellow dots at the cell periphery in merged image). To achieve higher resolution (~100 nm), a N-SIM/N-STORM super resolution microscope was used. Figure 3R2. Given that the necessary molecular components of SOCE/ROCE  as well as IP3R are present in pacemaker cells, we next examined if SOCE can be modulated by IP3R activation.We have previously reported that pacemaker cells express Type II IP3R Ca2+ signaling modulated pacemaker firing rate, it remains unknown whether IP3R agonists and antagonists (which can modulate heart rate) also modulate SOCE activity in SAN. Previous work on cardiac myocytes showed that overexpression of TRPC3 enhanced Ca2+ entry through SOCE while knock down of TRPCs inhibited SOCE , reintroduction of Ca2+ (1.8 mmol/L) caused a significant Ca2+ influx  as shown by an increase in Indo-1 ratio  in the presence of ET-1, a representative trace of which is shown in Figure 3 analog, IP3-BM (10 μmol/L) was also able to increase SOCE by 31 ± 10% . To test whether ET-1 or IP3-BM acted by opening IP3R release channels and thereby causing a greater store depletion (and hence SOCE), we examined the effect of enhancing store depletion by increasing both the CPA concentration and the incubation time in Ca2+ free solution. When the CPA concentration was increased from 10 μmol/L to 20 μmol/L and the incubation time in Ca2+ free buffer increased from 15 min to 30 min ET-1 was no longer able to further increase SOCE . Thus, 2-APB does not serve as a classic SOCE channel blocker in pacemaker cells, but can inhibit SOCE by a slower mechanism which may involve IP3R2 regulation as suggested previously  and/or OAG (100 μM), the agonists known to activate ROCE  ; the Ca2+ transient also increased by 26.1 ± 5.6%  and was associated with a 13.9% increase in pacemaker firing rate  in response to OAG treatment. There no significant changes by OAG in TRPC3 KO groups (n = 4) as shown in Figure 2+ entry through ROCE upon GPCR activation. 2 μM Pyr10 significantly reduced the resting Ca2+ elevation produced by OAG treatment in WT but not in TRPC3 KO mice  of PyR10 beyond TRPC3. In support of the latter idea, 20 μM Pyr10 reduced the amplitude of the Ca2+ transient in TRPC3 KO mice without significantly changing pacemaker firing rate or resting Ca2+ level (data not show).Figure . Figure  ThereforG Figure . Restingn Figure . Furthere Figure . Unexpec−/− mice). This suggests that TRPC3 channels may be involved in Ang II induced changes in the electrical properties of sinoatrial tissue. Recent evidence has implicated Ca2+ entry through TRPC3 as a pro-arrhythmic pathway  have also been implicated in failure of pacemaker function and cardiac arrhythmias   and IP3R2 antagonists (2-APB) through mainly their effect on increasing Ca2+ release and hence reducing store content . IP3R expression has been shown to increase in heart failure . While we found that Orai1 is expressed in mouse pacemaker cells (not shown), TRPC3 was more dominant at both mRNA and protein levels (Ju et al., We showed that TRPC3 expressing pacemaker cells also express the ER-CaCELewis, . HoweverCELewis, . In addif. In addition, it would be desirable to examine if other molecular components, such as NCX, SERCA, etc. that regulate intracellular Ca2+ are changed in TRPC3−/− mice. Despite these limitations, we shown that Ca2+ entry through both SOCE and ROCE occurs in murine SAN and appears to be regulated by the GPCRs in a GPCR≫ PLC ≫ IP3R/DAG ≫ Ca2+ signal transduction cascade. We suggest that TRPC3 channels might provide a potential target for future treatment of SAN dysfunction and AF as it links both receptor activation and intracellular Ca2+ signaling.The current study does not rule out the possibility that Pyr10 has effects on currents other than TRPC3, such as IThe 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.


Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway.Calcium ion (Ca Cadmium (Cd) is a non-essential toxic heavy metal widely spreaded in terrestrial and aquatic environments during mining and manufacturing. Cd accumulates in organisms by feeding and metabolic processes, whose toxicity is exemplified by an extremely long biological half-life within organisms 15–30 years), resulting in both acute and chronic toxicity. Acute exposure of animals to high dose of Cd can cause cell apoptosis within a short time , while l0 years, 2+) is a intracellular second messenger and its overload or disturbance in its intracellular compartmentalization can trigger apoptosis in various cells types [2+ concentration ([Ca2+]i) can be elicited through two pathways: i) the Ca2+ release from intracellular stores, mainly the endoplasmic reticulum (ER) and Golgi apparatus, or ii) the entry from the extracellular milieu [3 induction and Ca2+ release from ER and Golgi in primary murine macrophages. Intracellular Ca2+ increase involves the opening of the plasma membrane Ca2+ channels. Yeh et al. [2+]i increase in Madin Darby canine kidney cells via evoking Ca2+ entry through non-selective Ca2+ channels. Cd exposure led to the increase of [Ca2+]i. Extracellular Ca2+ removal by EGTA also diminished Cd-induced [Ca2+]i overloading but showed slight elevation of [Ca2+]i, suggesting that Cd-induced extracellular Ca2+ influx is an important source for elevated [Ca2+]i, but that another source of intracellular Ca2+ storage is also important i is usually regulated by Ca2+-ATPase, which can be activated by a small increase in [Ca2+] and export Ca2+ from the cytosol to the extracellular environment. Studies have shown that Cd disrupts intracellular Ca2+ homeostasis, leading to apoptosis in various types of cells , the gill cells were isolated from the untreated crab gill and were loaded with 5 μM Fluo-3/AM for 30 min at 37°C in the dark. After incubation, the cell suspensions were washed twice with PBS to remove the extracellular Fluo-3/AM. Then the cells were treated with 5.8 mg•L-1 CdCl2 for the different times. Fluorescent probe was excited at 488-nm, and emission fluorescent was filtered by a 525-nm filter. The images were recorded under a laser confocal microscope .To measure  level alteration was performed with Cd treatment for 0 to 60 min. As shown in 2+] in control cells, intracellular [Ca2+] level in Cd-treated cells for 20 min started to increase significantly and reached its peak at 35 min. The [Ca2+]i at 60 min was similar to that of control cells.To investigate whether the Cd-induced apoptosis is associated with [Ca2+]i in gill cells of S.henanense. To investigate the effects of Cd on CaM, the CaM content in the Cd signaling were measured using ELISA method. As shown in Cd treatment led to the changes of  and induce apoptosis in gill cells. We proposed that the change of [Ca2+] is involved in the Cd-induced cytotoxicity. To establish a link between Ca2+ and apoptosis in Cd signaling, crabs were treated with Ca2+ inhibitor (EGTA) for 4 h prior to Cd treatment. As shown in S. henanense. Pretreatment of Ca2+ inhibitor blocked the DNA fragmentation, indicating that Cd-elevated [Ca2+] is required for the apoptotic DNA fragmentation induced by Cd.Cd could lead to the increase of i level in PC12 and SH-SY5Y cells as well as primary murine neurons in a concentration- and time-dependent manner, which lasted for 24 h. Liu et al. [2+]i, which occurred as early as 0.5 h following treatment with Cd (100 μM), and cell apoptosis in thyroid cancer cells. In the present study, we found that Cd exposure altered the balance of intracellular Ca2+ and resulted in the increase of [Ca2+]i, which is consistent with the observations by Kim and Sharma [2+]i elevation and high concentration Cd  resulted in rapid and high-amplitude [Ca2+]i elevation. We treated the gill cells of crab with 5.8 mg•L-1 Cd  and found that [Ca2+] started to increase within 20 min and the increase started to drop after 40 min  . Chen et (300 s)  have fouu et al.  reportedd Sharma  who demor 40 min .2+-binding proteins as a calcium sensor and signal transducer to modulate cellular processes i elevation, and Cd-elevated [Ca2+]i activates MAPK and CaMKII, which are activated in the presence of Ca2+ and CaM  after treatments is carried out by energy-consuming Ca2+-ATPases and Na+/Ca2+-exchange mechanisms  after a Ca2+ signaling event. Ca2+-pumps might also contribute to the generation/modulation of Ca2+ signals [2+]  protects cells from apoptosis induced by Cd. This finding is in keeping with the results by other researchers [2+] elevation leading to cell apoptosis in J774A.1 (murine macrophages), PC12 (rat pheochromocytoma) and SH-SY5Y (human neuroblastoma) cell lines, and mouse thymocytes. Calcium ions are the most powerful inductors and mediators of cell death. Ca2+ signal is involved in the regulation of cell apoptosis induced by Cd in S. henanense. However, Ca2+ cross-talks with a variety of signaling pathways and the integrative function of the Ca2+ signaling in apoptosis requires further investigations.Cd can mediate a wide variety of cytotoxic and metabolic effects, such as altering the activities of various enzymes, interfering with the normal protective actions of essential metals, inducing oxidative stress, inhibiting mitochondrial ATP production, and altering gene expression that may trigger cell death by either apoptosis or necrosis . Cd led enanense ,37,38 anactivity . H2O2 prpoptosis . Howeverearchers ,26,70, w2+]i level and Ca2+-ATPase activity as well as the protein concentration of CaM, which is followed by gill cell apoptosis which was evidenced by activation of caspases- 3, -8 and -9, apoptotic DNA fragmentation, and apoptotic ultrastructure morphology. All the apoptotic features in the gill cells induced by Cd were inhibited by pretreatment of the animals with Ca2+ chelator EGTA. Thus, it is indicated that Cd elicits gill cell apoptosis through Ca2+ signal transduction pathway in the animal model.Our results showed that acute Cd exposure increased [Ca


We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar The resulting increase in cytosolic [Ca2+]i is essential for platelet degranulation via fusion of vesicles with the plasma membrane as well as rearrangement of actin filaments and therefore crucial for platelet shape change and aggregation.Cytosolic free calcium ions i provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Several fluorescent dyes have been developed to record [Ca2+]i dynamics in isolated platelets using cuvette-based methods: In contrast to the fluorescent Ca2+ indicator Quin-2 i in platelets and steadily displaces fluorimetric cuvette measurements [2+can be separated from [Ca2+]i of other cells like leukocytes, thereby also allowing experiments in whole blood, (ii) resting platelets can be distinguished from platelet aggregates,and (iii) high numbers of single cellular events are recorded, providing a high statistical power.Flow cytometry has become increasingly important for the measurement of [Caurements  as it of2+]i measurements are performed with Indo-1 labelled platelets employing UV-lasers , which is not a standard laser on most flow cytometers i measurement. Both indicators can be excited simultaneously by a standard blue argon laser and can be recorded in detector channels specific for green (Fluo-3/Fluo-4 emission 488nm) and red (Fura Red emission 637nm) light. Of note, Fluo-3/Fura Red has been shown to be more sensitive compared to Indo-1 i measurement of Fluo-4 and Fura Red labelled cells has been described due to the lack of appropriate calibration, as both dyes have dissimilar binding constants  for Ca2+. Applicable calibrations have so far only been published for single ratiometric dyes like Indo-1 and Fura-2 i in platelets. We describe the mathematical derivation to calibrate [Ca2+]I using Fluo-4 and Fura Red in a “two kd model” and confirm that this novel method is suitable for [Ca2+]i measurements in platelets. Furthermore, we show that Fluo-4/Fura Red dyes display an extended dynamic range and that the obtained results are unaffected by the formation of platelet aggregates.In this work, we provide a rapid protocol to quantify i = c(r) (cellular cytosolic Ca2+ concentration as a function of fluorescence ratio), and Equation 5 would be [Ca2+] as a function of ratio [c(r)], respectively:If Fluo-4 and Fura Red were considered as one single dye with two different emission wavelengths in FL1 and FL3 (like Indo-1), Equation This equation has been published previously for Fura-2 and has widely been applied also for Indo-1 measurements .2+ (B1 and B2) as a function of free [Ca2+] = c1, kd2, Bmax1, Bmax2…mass action binding constants and Bmax values for Ca2+ of dye-1 and dye-2, respectively.Equation min, FL1max, FL3min and FL3max in Equation This is transformed to fluorescences FL1(c) and FL3(c) given the minimum and maximum fluorescences of both dyes: FL1The ratio, defined in Rearranging 2+]i on the fluorescence ratio r(c) turned out to be the solution of an implicit quadratic equation. This equation had the additional constant parameters Rmin, Rmax, FL3min and FL3max that had to be obtained from FL1 and FL3 fluorescent data of Ca2+-free (Rmin and FL3max) and maximum Ca2+ standard samples (Rmax and FL3min), respectively. Kd1, kd2 were the apparent cellular kd values of the two dyes that were determined as follows:The newly derived mathematical equations for a true “double kd” calibration function are given in the Methods section. The dependence of i concentration was equilibrated with EGTA-containing Ca2+-buffers of known free Ca2+ concentrations(0 nM—12000 nM) in the presence of ionomycin, CCCP and nigericin. The changes of intraplatelet fluorescence of Fluo-4 and Fura Red at different Ca2+ concentrations are depicted in First, we determined the fluorescence range of Fluo-4/Fura Red-labelled platelets at different Ca7A and 7B yielding kd1 = 1069 nM for Fluo-4 and kd2 = 330 nM for Fura Red.Fitted curves in 1 and kd2 starting values were used as initial estimates for fitting Equation 10 (“two kd model”) to the ratio/concentration data pairs  with the obtained data points.These improved kd4, yielding a “combined kd” of 2208 nM), which showed less correlation to the observed data (26% higher standard error estimate) compared to the “double kd” model depicted in In 2+]i extends the linear range of the measurement. At all [Ca2+]i concentrations tested, the ratio FL1/FL3 showed a better linear relationship (r2 = 0.9973) to [Ca2+]i as solely FL1 (r2 = 0.9707) or FL3 (r2 = 0.9779) (data not shown). Moreover, the dynamic range  of Fluo-4 or Fura Red alone, which was 11.6 and 3.6, respectively, increased to 28.5 when the Fluo-4/Fura Red ratio was employed.We further show that ratiometric measurement of i . In cases where formation of platelet-platelet aggregates occurs during time-dependent flow cytometry, the acquisition of fluorescence ratios would therefore prevent misinterpretation of measured parameters that derive from changes in event size.From these data we conclude that formation of platelet aggregates affects single channel fluorescence even under constant i before, during and after stimulation of platelets with 1, 10 and 100 μM ADP  or whole blood (B) and only 2 calibration measurements in control buffers are required to set the minimal and the maximal calcium signal to calculate [Ca2+]i in any sample by the algorithm given in 2+]i dynamics by ratiometric flow cytometry using Fluo-4 and Fura Red. We provide equations to easily calculate [Ca2+]i levels in a 2-kd model as well as a ready-to-use protocol to quantify intraplatelet [Ca2+]i dynamics in whole blood and PRP by flow cytometry.In this study we describe a novel method to quantify intraplatelet [Ca2+]i can prevent such artefacts and the obtained results appear to be independent of cell/aggregate size and are therefore unaffected by platelet aggregate formation. Moreover we show that physiological concentrations of divalent ions, like magnesium (Mg2), zinc (Zn2+) and manganese (Mn2+), which also bind to Fluo-4 and Fura Red, do not significantly interfere with [Ca2+]i measurement  or decrease (Fura Red) in fluorescence upon binding ofi dynamics were only described for Indo-1, a dye that does not allow experiments with compounds that display a disturbing fluorescence emission in response to UV-light . In addition, Indo-1 shows a spectral overlap with NADH/NADPH autofluorescence and is therefore affected by variations in energy metabolism—furthermore, it bleaches more rapidly compared to Fluo-4 and Fura Red.Previously published ratiometric methods to quantify [Ca2+]i dynamics by flow cytometry, confocal microscopy and microplate screening applicationsi due to the presence of residual (non Ca2+ responsive) AM-esters, which associate with the membrane and/or were not washed away [Calcium-sensitive dyes are usually applied as membrane-permeable acetoxy-methyl ester derivatives (AM-esters). Cellular loading and accumulation of these dyes results from cleavage of these esters by unspecific intracellular esterases that convert the lipophilic (pro)dyes into polar membrane-impermeable forms that thereby also acquire the ability to bind Cahed away .2+]i dynamics.AM esters neutralize the charges of fluorescent dyes and indicators, allowing passive diffusion into live cells. The indicators are then converted by intracellular esterases into a cell-impermeant dye that is retained inside the cell.AM esters also enter organelles. While there are no significant differences between Fluo-3 and Fluo-4 in intracellular accumulation, compartimentalisation is higher in Fluo-4 compared to Fluo-3. Moreover, Fluo-4 shows a higher accumulation in the nucleus compared to Fluo-3 , but sin2+]i as well as for intraplatelet [Ca2+]i dynamics.We are able to show that application of Fluo-4/Fura Red ratio is suitable for measurement of basal intraplatelet [Ca2+]i depends on experimental conditions and furthermore might differ between individual donors. Therefore, to quantify [Ca2+]i dynamics calibration with only 2 buffers, a calcium free and a high calcium (10 mM CaEGTA) buffer, is necessary. As active calcium transport processes might impose gradients or affect equilibration, CCCP needs to be added as a metabolic inhibitor of mitochondria in a glucose-free buffer i dynamics in whole blood or PRP, which yields results that are unaffected by platelet aggregate formation using flow cytometry. This method can potentially be extended to other applications like live cell microscopy under flow conditions. Further research is necessary to determine if the described kds are also suitable for flow-based immunofluorescence microscopy.Taken together we describe a novel, rapid method to quantify intraplatelet [CaS1 File(DOCX)Click here for additional data file.


RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog  S. cerevisiae as a model organism, here we show that Rad9 oligomers block the removal of hypo-active Mre11 protein from a persistent DSB, thus limiting initiation of resection and the recruitment of the recombination factor Rad52, in the absence of Sae2. Altogether, these findings pinpoint a critical role of 53BP1/Rad9 in balancing HR and NHEJ repair events throughout the cell cycle.DNA double strand breaks (DSBs) are among the most deleterious types of damage occurring in the genome, as failure to repair these lesions through either non-homologous-end-joining (NHEJ) or homologous recombination (HR) leads to genetic instability. The 5′ strand of a DSB can be nucleolytically degraded by several nucleases and associated factors, including Mre11, CtIP/Sae2, Exo1 and Dna2 together with Bloom helicase/Sgs1, through a finely regulated process called DSB resection. Once resection is initiated, error-prone NHEJ is prevented. Several findings suggest that DSB resection is a double-edged sword, if not finely regulated, since on one hand it is needed for faithful HR, but on the other it may lead to extensive DNA deletions associated with genome instability. Both in mammals and yeast, 53BP1/Rad9 protein binds near the lesion and counteracts the resection process, limiting the formation of ssDNA. By using  S. cerevisiae the ends of a double-strand DNA break (DSB) are recognized and bound by the Mre11-Rad50-Xrs2 (MRX) complex and the Ku70-Ku80 heterodimer, which compete for end binding. Once the MRX complex, together with CDK1-phosphorylated Sae2 (CtIP in human), initiates resection of the DNA ends, Ku70-Ku80 binding and NHEJ (non-homologous end-joining) are prevented Similarly to what is seen in higher eukaryotes, in The regulation of DSB end resection is very important to choose the right pathway to repair a DSB and avoid chromosomal rearrangements It is now clear that the DNA damage checkpoint response (DDR) plays a central role in regulating DSB end resection. In fact, while resection proceeds, the formation of RPA-coated ssDNA activates the upstream kinase Mec1  and the effector kinase Rad53 , which in turn phosphorylates and inhibits Exo1 RAD9 was originally identified as the first checkpoint gene in S. cerevisiae and recognized as an “adaptor” protein, linking the upstream kinase Mec1 to the activation of effector kinases Rad53 and Chk1. Rad9 is recruited to chromatin through three different pathways: i) the constitutive interaction with the histone H3 methylated at the K79 residue by Dot1 Moreover, studies both in yeast and mammals showed that Exo1 and other DNA end-processing enzymes are inhibited through a physical structural “barrier” formed by Rad9 oligomers  bound near a DSB Notably, the Rad9-mediated inhibition of DSB resection is a regulatory function conserved throughout evolution. In fact, 53BP1 facilitates NHEJ at the expense of HR, protecting DNA ends from inappropriate 5' resection, in cooperation with the telomere binding protein RIF1 Here, we show that in the absence of Sae2, or in presence of mutations affecting Mre11 nuclease activity, Rad9 dimers and/or oligomers, recruited near a DSB mainly by Dpb11 interaction, inhibit the short-range DNA end processing, thereby preventing Mre11 removal from the lesion and limiting Rad52 recruitment by an Sgs1-dependent mechanism. As a consequence, DSB ends cannot be kept efficiently tethered to each other, and repair through an SSA process is prevented. We propose a novel molecular role of Rad9/53BP1 to protect genome integrity from extensive DNA degradation and rearrangements during DSB repair, also suggesting important implications for malignant transformation in mammalian cells.RAD9 gene in yeast leads to faster DSB resection and repair through an SSA process RAD9 gene with mutations in genes encoding factors either involved in the short-range (SAE2), or the long-range  DSB resection leu2 repeats one of which is 25kb from the DSB RAD9, EXO1, SGS1 and SAE2 to obtain all viable single, double and triple mutant combinations. Although the sae2Δ sgs1Δ double mutant is a synthetic lethal combination rad9Δ interestingly suppresses sae2Δ sgs1Δ lethality . This is a common genetic strategy to induce the degradation of a protein by the addition of auxin compound to the cell culture medium DNA2. By Southern blotting analysis, we found that the sae2Δ rad9Δ double mutant cells do not repair a DSB in the absence of Dna2  DNA is not biased by a repair process A critical step to repair a DSB through SSA is 5′ to 3′ resection of the DSB end. Therefore, based on our results in SAE2, EXO1 and SGS1 genes, and it is severely compromised in the exo1Δ sgs1Δ double mutant exo1Δ sgs1Δ rad9Δ triple mutant  protein variant, which does not interact with Dpb11 rad9-2A mutation increase the viability of sae2Δ cells after one DSB, with the major contribution done by the mutation that abrogates the Rad9-Dpb11 interaction  was calculated on three independent experiments.ChIP analysis was performed as described previously RsaI restriction enzime (NEB) that cuts inside the amplicons at 0.15 kb and 4.8 kb from the DSB, but not in the PRE1 control region on chromosome V. qPCR was performed on both digested and undigested templates using StoS Quantitative Master Mix 2X SYBR Green (Genespin) with the Biorad MyIQ2 PCR system. The ssDNA percentage over total DNA was calculated using the following formula: % ssDNA  =  {100/[(1+2CtΔ)/2]}/f, in which ΔCt values are the difference in average cycles between digested template and undigested template of a given time point and f is the HO cut efficiency measured by Southern blot analysis.Quantitative PCR (qPCR) analysis of DSB resection was performed accordingly to 4 was added one hour before inducing HO cut with galactose, accordingly to Cells of strains derivative from yJK40.6 background were grown in YEP+raf and blocked 3 hours in G2 with nocodazole. 160 µM CuSOS1 FigRAD9 rescues the lethality of the sae2Δ cells after a DSB through the helicase activity of Sgs1. (A) Meiotic tetrads from the indicated cross were dissected on YEPD plates that were incubated at 25°C, following by spores genotyping. (B) A plasmid vector expressing either the wild type or sgs1-K706A allele of SGS1 gene was inserted by transformation into the YMV80 derivative sae2Δ sgs1Δ rad9Δ triple mutant. For each YMV80 derivative strain indicated in the Figure, the number of colonies grown after 3 days at 28°C in YEP+gal was normalized respect YEP+glu. Plotted values are the mean values ± SD from three independent experiments.Deletion of (TIF)Click here for additional data file.S2 FigRAD9 rescues DSB repair defects of sae2Δ cells through a Rad51-independent pathway. (A) Exponentially growing cell cultures of the wild type YMV80 strain and the indicated derivatives were serially diluted (1∶10), and each dilution was spotted out into YEP+Raf or YEP+Raf+Gal plates. Plates were incubated 3 days at 28°C. (B) Exponentially growing YEP+raf cell cultures of the wild type YMV80 strain and the indicated derivatives were synchronized and kept blocked in G2/M phase with nocodazole treatment; galactose was added at time zero to induce HO-cut. Genomic DNA, extracted from samples taken at the indicated times, was analyzed for DSB formation and repair, as described in Deletion of (TIF)Click here for additional data file.S3 FigMAT locus and expressing the HO nuclease under GAL1 promoter, were synchronized and kept in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Genomic DNA, extracted from samples taken at the indicated times, was analyzed for ssDNA formation, as described in Rad9 limits an Sgs1- and Exo1- dependent DSB resection. (A) Exponentially growing YEP+raf cell cultures of the wild type JKM139 strain and the indicated derivatives, carrying a unique HO cut site at (TIF)Click here for additional data file.S4 Figdot1Δ derivative strains. (A) Exponentially growing YEP+raf cell cultures of the wild type JKM139 strain and the indicated derivatives, carrying a unique HO cut site at MAT locus and expressing the HO nuclease under GAL1 promoter, were synchronized and kept in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Genomic DNA, extracted from samples taken at the indicated times, was analyzed for ssDNA formation, as described in sae2Δ blots are the same used in Analysis of DSB resection in (TIF)Click here for additional data file.S5 FigAnalysis of Rad9 binding near a DSB. Cells of the wild type JKM139 strain and the indicated derivatives, expressing a Rad9-3HA fusion protein, were grown in YEP+raf and synchronized in G2/M phases by nocodazole treatment. Galactose was added at time 0 to induce HO. Relative fold enrichment of Rad9-3HA at 0.1 kb from the HO cleavage site was evaluated after ChIP with anti-HA antibodies and qPCR analysis. Plotted values are the mean values ± SEM from three independent experiments.(TIF)Click here for additional data file.S6 Figrad9-ΔBRCT-FKBP derivative were grown in YEP+raf. Cell cultures were split in two and one half was treated with AP20187 for 1 hr, before adding galactose to induce HO. Cells have been taken at the indicated times and protein extracts were done. Rad9 and Rad53 were detected by western blotting.Analysis of the expression levels and phosphorylation of various Rad9 protein variants. (A) Cells of the wild type YMV80 strain and the indicated derivatives were grown in YEP+raf. Galactose was added at time 0 to induce HO. Cells have been taken at the indicated times and protein extracts were done. Rad9 and Rad53 were detected by western blotting. (B) Cells of the wild type YMV80 strain and the (TIF)Click here for additional data file.S7 FigKU70 does not rescue viability of YMV80 derivative sae2Δ cells, following a DSB. Viability of the wild type YMV80 strain and the indicated derivatives, plated on YEP+gal. For each strain, the number of colonies grown after 3 days at 28°C in YEP+gal was normalized respect YEP+glu. Plotted values are the mean values ± SD from three independent experiments.Deletion of (TIF)Click here for additional data file.S1 TableList of yeast strains described in this work.(DOCX)Click here for additional data file.S2 TableList of the oligonucleotides used for ChIP and DSB resection analyses.(DOCX)Click here for additional data file.


This discovery provided an important missing piece in our understanding of the pathomechanism of filter barrier damage, revealing Ca2+ signaling as critical for podocyte health and disease. The identification of the two Ca2+ permeant channels TRPC5 and TRPC6 as mediators of this pathway not only bolstered the importance of podocyte cytoskeleton dynamics but also revealed promising drug targets for treatment-resistant nephrotic syndrome. This review will focus on this novel signaling pathway in primary podocytopathies and its implications for next-generation therapies for glomerular disease.With more than 6,000 new pediatric patients with treatment-resistant nephrotic syndrome in the US each year alone, the unmet need for novel, podocyte-specific therapies is substantial. Recently, the established therapeutic benefit of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) was used as a starting point to gain insight into the pathomechanism of primary podocytopathies. A calcium (Ca A 16-year-old boy who presented with nephrotic syndrome since the age of 7, with an initial biopsy showing minimal change disease (MCD), and with a subsequent biopsy showing primary FSGS  at age 9, presented with relapsing steroid-resistant nephrotic syndrome. The patient had preserved renal function with an average serum creatinine 0.5 mg/dl. His medications included lisinopril 10 mg daily, simvastatin 40 mg daily, and furosemide as needed, titrated to symptoms. Cyclosporine A, cytotoxic therapy with cyclophosphamide, mycophenolate mofetil, and rituximab had been added to steroids at different times during his adolescent years, without success in achieving remission. At age 16, while in partial remission on high-dose oral steroids, and during a subsequent attempt to slowly taper the steroids, he experienced recurrence of proteinuria with a urinary protein/creatinine ratio (UPC) of 12. Multiple attempts to achieve remission with high-dose steroids were unsuccessful. A few weeks later, he was admitted to the hospital with anasarca that required intravenous diuretics. A repeat kidney biopsy confirmed the diagnosis of primary podocytopathy or FSGS. What further therapeutic interventions may be available to this patient with steroid-resistant nephrotic syndrome?One of the main tasks of the kidney is to deplete low molecular weight molecules from the plasma, while larger, vital proteins such as albumin are retained in the bloodstream . The gloBased on the underlying disease mechanism, we would like to reinforce a podocentric classification that distinguishes primary and secondary podocytopathies. While secondary podocytopathies are the consequence of well-defined inflammatory responses in a variety of immune-mediated glomerular diseases such as ANCA vasculitis, lupus nephritis, or other immune complex glomerulonephritides, where the podocyte may be “collateral damage” due to primary endothelial and immune complex-mediated injury, primary podocytopathies are caused, by definition, by podocyte-specific injury. Histologically, primary podocytopathies are a heterogeneous group of kidney diseases with a great variety of anatomical features. These range from profound structural changes in the podocyte cytoskeleton only detectable at the electron microscopic level ) all the way to profound light microscopically detectable focal sclerosis (as in FSGS) and/or completely collapsed glomeruli (as in collapsing glomerulonephritis) . This reThe filtration barrier is composed of the fenestrated endothelium of the glomerular vessels, the glomerular basement membrane (GBM), and the interdigitating podocyte foot processes (FPs). Podocyte structures called slit diaphragms (SD) function like modified adherens junctions connecting neighboring FPs . Through2+-mediated pathway connects the angiotensin type 1 receptor (AT1R) to podocyte cytoskeletal dynamics. This pathway indeed provided an explanation for an important missing piece in our understanding of the pathomechanism of filter barrier damage, revealing Ca2+, a central player of the intracellular signaling apparatus in any cell type, as critical for podocyte health and disease. This review will focus on this novel signaling pathway and its implications for next generation therapies for glomerular disease.Understanding the importance of the podocyte cytoskeleton for a functioning renal filter barrier poses a fundamental question: What is the underlying mechanism of cytoskeletal rearrangements in podocytes? Starting from the established benefit of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB) as our first-line, standard-of-care therapy for glomerular disease , and ask2+ is a vital second messenger in virtually every cell type [2+ signaling relies on a low intracellular Ca2+ resting concentration [Ca2+]i (0.1 μM), roughly four orders of magnitude below the extracellular Ca2+ concentration [Ca2+]e (2 mM). A Ca2+ signal is induced by an increase of the [Ca2+]i, whereby the major sources of Ca2+ are the extracellular space and intracellular Ca2+ stores such as the endoplasmic reticulum (ER) and the mitochondria [2+ buffers constitute the cellular Ca2+ regulatory apparatus. Their well-coordinated interplay allows both the induction of intracellular Ca2+ signals (ON mechanisms) and the relaxation of the system towards its vital low resting [Ca2+]i (OFF mechanisms) i, Ca2+ binds to highly conserved Ca2+ binding domains, such as EF-hand domains i induces cell death i, sufficient to activate a localized effector [However, how can individual TRPC channels mediate distinct cellular events? A unifying explanation can be found in the existence of Caeffector . Thus, beffector . Intereseffector  and proteffector , indicateffector . In summeffector .2+-dependent phosphatase calcineurin is typically known for activating the transcription factor nuclear factor of activated T-cell (NFAT) in T-cells, upregulating interleukin 2 (IL-2), and thus inducing the T-cell response [The Caresponse . Howeverresponse , which presponse . Activatresponse . This firesponse , revealiThe fact that FSGS-causing TRPC6 gain-of-function mutations activate calcineurin-NFAT-dependent gene transcription  further How do the experimental findings summarized here translate into clinical medicine? One way to evaluate the significance of these insights is to look at the consequences of mutations in key molecules of the proposed signaling pathway. Mutations of TRP channels have been associated with a number of different diseases. Such TRP channelopathies include night blindness (TRPM1) , progres2+ channel causing FSGS initially suggested the potential for a new pathomechanism [2+-mediated pathways may be excellent therapeutic targets for a disease that currently lacks effective treatment options.At that time, proteinuric kidney disease was primarily associated with rearrangements in the cytoskeletal structure of podocytes, thus the identification of a Caechanism . Howeverechanism . Just reechanism . The conechanism . This st2+ channel blocker), lidocaine (voltage-gated Na+ channel blocker) and diazepam  [Drugs targeting ion channels are in widespread clinical use: common representatives are verapamil (voltage-gated L-type Cahanne6l) . Historihanne6l) . Howeverhanne6l) . Modern 2+ physiology in podocytes (TRPC5 and TRPC6), we may now be poised to uncover new treatment options for primary podocytopathies based on the modulation of TRPC5 and TRPC6 channel activity.Given the orthogonal convergence of human genetics (TRPC6), the cell biology of the actin cytoskeleton (RhoGTPases) and our new knowledge of Ca2+ homeostasis in podocytes. Indeed, the small molecule inhibitor ML204 was not only shown to be an effective pore blocker of TRPC5-mediated Ca2+ transients in podocytes, but it was also shown to rescue podocytes from disease-associated cytoskeletal remodeling. This effect was further validated with Ca2+ imaging in isolated glomeruli, emphasizing the physiological relevance of the in vitro model in podocytes [A first promising step in this direction was the identification of ML204, a small molecule inhibitor of TRPC4/5 channels . Since Todocytes . These d2+-mediated toxicity and cell death [2+ influx.The finding that gain-of-function mutations in TRPC6 cause familial FSGS  has alsoll death –55). The2+-regulated phosphatase calcineurin as an important link between TRPC/Ca2+ signaling and cytoskeletal dynamics in podocytes [Identification of the Caodocytes , 28 introdocytes . Consideodocytes . SpecifiThe emerging importance of the essential role of an intact podocyte cytoskeleton for a functioning filtration barrier has been further bolstered by the identification of B7-1 (CD80), as yet another potentially druggable component of the regulatory apparatus of the podocyte cytoskeleton. B7-1 was shown to be upregulated upon podocyte injury , thus pr2+ homeostasis, because the consequence of excess Ca2+ is cytoskeletal collapse [The hallmark of primary podocytopathies is proteinuria, a direct consequence of filter barrier damage. Functionally, podocytes are critical for maintaining an intact filtration barrier . Importacollapse , 52–55 wcollapse .2+-mediated cytoskeletal rearrangements in podocytes, and the notion of maintaining a proper balance between homeostatic TRPC6/RhoA versus inducible TRPC5/Rac1 activity constitutes a novel paradigm for understanding filter barrier damage in proteinuric diseases. Together with the development of improved TRPC channel inhibitors, these findings constitute a new approach to the unmet need of effective treatment options for primary podocytopathies such as MCD, FSGS, and diabetic kidney disease, as well as other acquired proteinuric kidney diseases. Furthermore, new drugs interacting with downstream targets of calcineurin, most importantly synaptopodin, could constitute a treatment option that is, due to an optimized side effect spectrum, more attractive than the currently used CsA  to a decompensated state of permanent podocyte loss and replacement with scar (FSGS). This new, molecularly-defined understanding of the mechanisms underlying primary podocytopathies also brings into crisper focus the therapeutic potential of targeting the podocyte cytoskeleton.With more than 6,000 new pediatric patients suffering from treatment-resistant nephrotic syndrome in the US each year alone , the unm


Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca2+-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases.Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl Free ionized extracellular calcium concentration  and physiological (Ca2+o) CaSR activation on fluid secretion and identified the main Cl− channels responsible for this process in chemically-defined, serum-free whole mouse and human lung explant cultures. Finally, the signalling machinery involved in CaSR-driven fluid secretion was examined using live imaging in an in vitro cell reporter system.In this study, we sought to examine how activation of the CaSR leads to increased fluid secretion and lung growth within the developing human lung. Because much of the work aimed at testing the contribution of each ClWild type C57Bl/6 mice were housed conventionally with a 12 h light:dark cycle and had free access to food and water. All animal procedures were approved by the UK Home Office and carried out in the UK in accordance with the Animal (Scientific Procedures) Act 1986.Human fetal tissue was obtained from ethically-consented maternal donor medical termination at 7–11 week of pregnancy following the guidelines of the Polkinghorne and Department of Health reports with approval from South East Wales Research Ethics Committee25Human fetal (7–11 week post conception) or E12.5 mouse lungs were fixed in formalin overnight and subsequently embedded in paraffin. 5 μm thick sections were deparaffinised in xylene and rehydrated using a decreasing alcohol-water series , followed by washing in distilled water. Fischer rat thyroid (FRT) cells stably transfected with CFTR were grown on sterilized glass coverslips of 13 mm diameter in 24 well plates. The culture medium was removed and the cells were fixed in 4% (w/v) PFA in PBS for 15 minutes.The immunohistochemical protocol used was established and optimised for each species and antibody see . Briefly4Cl in PBS to quench free aldehyde groups remaining from the fixation step. Permeabilization and/or heat-induced epitope retrieval was performed as described above. Non-specific antibody binding was prevented by incubating the slides with blocking solution for 1 hour at room temperature.5 μm thick, paraffin embedded week 10 human fetal lungs were dewaxed and rehydrated as described above. Samples for immunofluorescence were washed in 50 mM NHPrimary antibodies were diluted in blocking solution and applied to slides overnight at 4 °C. After washing in PBS, primary antibody binding was visualized using Alexa Fluor 594 fluorescence-dye coupled secondary antibodies in blocking buffer. Nuclei were counterstained with Hoechst 34580. Coverslips with adhering cells were then mounted on standard glass slides using ProLong Gold® (Life Technologies), while human lung slide were mounted in DPX after dehydration. Negative controls were carried out through substitution of the primary antibody with either rabbit or goat serum. Slides and cells were left to dry overnight and then epifluorescence images of immunostained tissues and cells were analysed and photographed with an Olympus BX61 automated microscope equipped with a 100 W high pressure mercury lamp, using AnalySIS software .Lungs explanted from 7–9 week human fetal tissue and E12.5 mice were cultured for 48–72 hours according to previous published protocols1027282+o conditions, [Ca2+]o in the DMEM-F12 medium was increased from 1.05 mM [Ca2+]o to 1.70 mM [Ca2+]o using 1 M CaCl2 . Inhibitors of chloride-transporting mechanisms included the wide-spectrum anion exchange inhibitor 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (Sigma-Aldrich) and the CFTR specific inhibitor Inh-172 (Tocris) and the loop diuretic, bumetanide (Sigma-Aldrich). All chloride channel inhibitors were dissolved in DMSO, which has previously been shown not to affect lung explant growthFor the fetal Ca2, 1 CaCl2, 5 HEPES, 10 glucose, pH 7.45. A 2–3 MΩ borosilicate glass electrode  was filled with a solution containing a 0.4% trypan blue/0.85% saline solution (Invitrogen). Electrodes were slowly pushed into a terminal lumen whilst maintaining positive pressure, with the presence of trypan blue in the lumen being indicative of successful access to the lumen. Once access to the lumen had been achieved, positive pressure was immediately removed and the PD was recorded after a 5 min equilibration period. E12.5 mouse lungs were cultured for 48 h in the presence of 1.05 mM or 1.70 mM Ca2+o in the presence or absence of the wide-spectrum anion exchange inhibitor which blocks Ca2+-activated chloride channels including TMEM16A and bestrophin, 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)1302+o control.Transepithelial electrical potential difference (PD), indicative of anion-driven fluid secretion into the lumen of the developing lung, was recorded as previously describedCaSR-dependent activation of CFTR was measured by single cell imaging of Fischer rat thyroid (FRT) cells stably transfected with the human CFTR channel and a halide sensitive YFP (YFP-H148). The FRT-CFTR-YFP cell line has previously been used for high-throughput screening for the identification of CFTR modulatorsFRT-CFTR-YFP cells were maintained in Coon’s modification of F12 (Sigma-Aldrich) supplemented with sodium bicarbonate (3.2 mM), 2 mM glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (Invitrogen). Cells were plated on to 13 mm glass coverslips 48 h before being placed in the light path of an inverted microscope . A Xenon arc fluorescence light source was used to excite the intracellular YFP at 470 nm and visualise emission at 520 nm.2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2 and 0.5 mM MgCl2, pH 7.4) in presence of pharmacological agents including the adenylate cyclase activator forskolin (20 μM), the specific CaSR allosteric activator NPS-R568 (1 μM), the CFTR inhibitor Inh-172 (10 μM) and the adenylate cyclase inhibitor MDL-12330A (25 μM). During the experiments cells were pre-incubated with Dulbecco’s PBS in the presence and absence of various activators and inhibitors for at least 5 min before being perfused with Iodide-rich Dulbecco’s PBS  while YFP fluorescence was imaged. Iodide influx quenches the cells YFP fluorescence with a rate that is dependent on the halide permeability of the cell, and therefore on the activity of the CFTR channel. Quenching rates were calculated by fitting the YFP fluorescence decay with a one-phase exponential decay function using GraphPad Prism 6.01 .Cells were equilibrated using standard Dulbecco’s PBS , bestrophin-1 and the cystic fibrosis transmembrane conductance regulator (CFTR). TMEM16A expression was detected apically in mouse fetal lungs  appeared to be comparable in morphology and transepithelial PD . DIDS did not significantly alter the transepithelial PD when added to medium containing 1.70 mM Ca2+o  as a surrogate for anion-driven fluid secretion. Thus, E12.5 mouse lungs were cultured in the presence of blockers of those Cl− entry by measuring transpetithelial PD in lungs cultured in the presence of the loop diuretic bumetanide, an NKCC1 blocker. Lungs cultured in medium containing 1.05 mM Ca2+o showed a decrease in luminal PD to increasing concentrations of bumetanide, with luminal PD reduced by ~50% compared to 1.05 mM Ca2+o alone  increased transepithelial PD compared to lungs cultured in the presence of medium containing 1.05 mM Ca2+o control . These results suggest that basolateral Cl− entry is not regulated by CaSR/Ca2+o.Furthermore, we tested the contribution that CaSR makes to the regulation of basolateral Cl−-secretion. To test this hypothesis further, we performed electrophysiological recordings of mouse and human lung explant cultured in the presence or absence of the specific CaSR allosteric activator2+o medium containing NPS-R568 (10 nM) led to a significant increase in transluminal PD from 100 ± 13% (n = 9) to 184 ± 19% ; and another comparing 1.05 mM Ca2+o + 100 nM R568 vs. 1.05 mM Ca2+o + 100 nM R568 + 10 μM Inh-172 (to test for CFTR-dependent CaSR-mediated activation of fluid secretion). In both sets of experiments, transepithelial PD was normalised against the 1.05 mM Ca2+o + 100 nM R568 condition. Human lung rudiments cultured in medium containing 1.05 mM Ca2+o showed significantly reduced transepithelial PD compared to those cultured in 1.05 mM Ca2+o + 100 nM R568 , before measurements of transepithelial PD were conducted. Lungs cultured for 48 hours in medium containing 1.70 mM Ca2+o exhibited the expected increase in luminal PD to 221 ± 17%  before addition of the iodide-rich PBS. Pre-incubation with NPS R-568 led to a significant increase in the rate of YFP quenching compared to control, quenching YFP fluorescence to 61 ± 3% at a rate constant of k = 1.31 ± 0.03 (2+i)-mobilising agonists are known to couple to Ca2+-activated chloride channels2+i-mobilising agonists activate CFTR by a mechanism involving Ca2+i-dependent activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling in human bronchial epithelial cells2+i mobilisation2+i, would be expected to activate AC1, an effect which would result in opening of CFTR.Classically CFTR is activated by cyclic AMP (cAMP)-dependent activation of protein kinase A (PKA). However, intracellular calcium ion . However, pharmacological approaches demonstrated that the calcimimetic NPS R-568 induced opening of CFTR that was dependent on adenylate cyclase. Of the nine G-protein–responsive transmembrane adenylyl cyclase (AC1-9) isoforms identified, AC1 can be stimulated by Ca2+i/calmodulin in contrast to many other ACs, which are either Ca2+-insensitive or Ca2+-inhibited. In addition, Namkung et al. have showed that, in human bronchial cells, the Ca2+i mobilising agonist, UTP, can stimulate CFTR opening through AC12+i mobilisation, we propose that, in the developing human lung epithelium, CaSR activation by NPS R-568 and/or fetal hypercalcemia leads to an increase in Ca2+i, attendant activation of AC1 and opening of CFTR. A potentially similar mechanism has been described in duodenal epithelial cells, where CaSR agonists induce bicarbonate secretion via CFTR, through activation of PLC and increase in Ca2+i Conventionally CFTR is activated by cAMP and PKA whereas Cain utero disruption of CFTR leads to progressive changes in lung function and structure that appear to predispose to adult lung disease58586062636465Newborns with CF were long considered to be born with normal lungs2+i, which in turn activates the Ca2+-stimulated AC1, inducing opening of CFTR within the apical membrane of the developing pulmonary epithelium .