NRAS and KRAS proto-oncogenes are among the most common in human cancer and frequently occur in acute myeloid leukemia (AML). An inducible NRAS(V12)-driven AML mouse model has established a critical role for continued NRAS(V12) expression in leukemia maintenance. In this model genetic suppression of NRAS(V12) expression results in rapid leukemia remission, but some mice undergo spontaneous relapse with NRAS(V12)-independent (NRI) AMLs providing an opportunity to identify mechanisms that bypass the requirement for Ras oncogene activity and drive leukemia relapse. We found that relapsed NRI AMLs are devoid of NRAS(V12) expression and signaling through the major oncogenic Ras effector pathways, phosphatidylinositol-3-kinase and mitogen-activated protein kinase, but express higher levels of an alternate Ras effector, Ralb, and exhibit NRI phosphorylation of the RALB effector TBK1, implicating RALB signaling in AML relapse. Functional studies confirmed that inhibiting CDK5-mediated RALB activation with a clinically relevant experimental drug, dinaciclib, led to potent RALB-dependent antileukemic effects in human AML cell lines, induced apoptosis in patient-derived AML samples in vitro and led to a 2-log reduction in the leukemic burden in patient-derived xenograft mice. Furthermore, dinaciclib potently suppressed the clonogenic potential of relapsed NRI AMLs in vitro and prevented the development of relapsed AML in vivo. Our findings demonstrate that Ras oncogene-independent activation of RALB signaling is a therapeutically targetable mechanism of escape from NRAS oncogene addiction in AML.Somatic mutations that lead to constitutive activation of  While the genetic landscape of AML has been extensively characterized, genetically based targeted therapies have yet to be realized and the optimal therapeutic target(s) are not known.3Despite aggressive combination chemotherapy, the majority of patients with acute myeloid leukemia (AML) die of relapsed treatment refractory disease.RAS proto-oncogenes are mutated in about 10–15% of human AML, and additional recurring AML mutations rely on Ras signaling for their oncogenic effects .6 A critical role for oncogenic Nras and Kras in leukemogenesis and maintenance of AML cells has been substantiated in genetically engineered mouse models.10 Furthermore, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways downstream of Ras have been shown to regulate leukemic stem cell self-renewal in AML.11 In fact, the MAPK and PI3K pathways are active in the majority of patient-derived AML samples, further supporting a key role for Ras signaling in AML maintenance.13RAS mutations are among the most common molecular alterations in human cancer, and thus Ras has been the focus of intense interest for drug development.14 A major obstacle for targeted cancer treatment approaches has been the almost ubiquitous development of treatment resistance. For example, disruption of the post-translational modification of Ras with farnasyltransferase inhibitors demonstrated encouraging preclinical activity, but their clinical activity has been limited owing to resistance conferred by alternative biochemical pathways for the prenylation of Ras.15 Targeting BRAF and/or MEK has shown encouraging responses for BRAF(V600E) mutant non-small-cell lung cancer and melanoma, but responses have been variable and transient owing to treatment resistance.20 It has become clear that diverse mechanisms such as disruption of drug–target interaction, mutations or amplifications that lead to activation of downstream signaling pathways, and/or activation of alternative growth and survival pathways can lead to resistance to most, if not all, targeted cancer therapies.22 Therefore, a better understanding of disease and context-specific resistance mechanisms will be essential to develop rational combination strategies for specific diseases.Oncogenic tetracycline-repressible NRAS(V12) and Mll-AF9-driven AML genetically engineered mouse model (tNM AML).8 The leukemia cells in this model are NRAS(V12)-dependent (NRD), and suppressing NRAS(V12) expression leads to rapid remission in leukemic mice, further highlighting the clinical potential of targeting oncogenic NRAS in AML.8 This model faithfully recapitulates the key challenge for clinically targeted cancer treatment, in that some mice spontaneously relapse with NRAS(V12)-independent (NRI) AML, providing a robust tool to study the mechanisms of relapse after Ras oncogene-targeted therapy. We interrogated key cancer signaling pathways, performed global gene expression analysis and performed functional studies to identify mechanisms that drive relapse with NRI AML and provide insight into the rational development of novel targeted treatment approaches for AML.To model Ras oncogene-targeted therapy for AML, we used a 8 In this system, suppression of NRAS(V12) expression resulted in rapid leukemia regression  in relapsed NRI AMLs in the presence of Dox treatment, thereby confirming the NRAS(V12) independence of the relapsed NRI AMLs (We confirmed leukemia . SimilarRI2 AMLs . WesternNRI AMLs .NRAS(V12) in both de novo NRD and relapsed NRI AML cells (NRAS(V12) suppression in de novo NRD AML, but was maintained at higher levels in relapsed NRI AMLs, even in the absence of NRAS(V12) expression  analyses of key cancer signaling pathways. Flow cytometric analysis confirmed a decrease in canonical oncogenic Ras effector signaling pathways—MAPK and PI3K—with decreased levels of phosphorylated ERK and AKT following Dox-mediated suppression of ML cells . Phosphopression . The exppression . Proapoppression , suggestpression .23 Of the 292 epitopes interrogated, 27 showed different levels of protein expression or phosphorylation between Dox-treated NRD AML and relapsed NRI AMLs .8 NRI1 and NRI2 AML cells were more similar to each other than NRD AML cells as determined by unsupervised clustering. Notably, NRI1 and NRI2 samples did not segregate in unsupervised clustering based on all identified transcripts . Bruton’s tyrosine kinase, a member of the Tec kinase family, has been identified as a targetable pathway in AML.28 The PI3K signaling pathway was the most inhibited pathway in relapsed NRI AMLs compared with de novo NRD AML , consistent with their decreased dependence on canonical Ras signaling. The canonical nuclear factor-κB signaling pathway was activated in relapsed NRI AMLs compared with de novo NRD AML  and NFKB1 was among the predicted activated upstream regulators in relapsed NRI AML cells . Other predicted upstream regulators that were activated in relapsed NRI AMLs included HOXA9 , CEBPA  and STAT3  that have established roles in AML.31We performed Ingenuity Pathway Analysis to identify enriched cellular processes, signaling pathways and predicted upstream regulators of differentially expressed genes . The Tecde novo NRD AML and relapsed NRI AMLs  independence for relapsed NRI AMLs we evaluated the differential expression of genes that are known to be associated with RALB activation and signaling 36 (AKT (myrAKT) or CRAF 22W, did not effectively rescue leukemic colony formation  inhibitors are lacking, dinaciclib, a cyclin-dependent kinase inhibitor, has been shown to inhibit CDK5-mediated activation of Ral signaling.P levels . DinacicP levels , but didll cycle . Dinacicll cycle . To veri(Q72L)36 . Notablyormation .37In vitro treatment of a panel of AML samples with dinaciclib uniformly resulted in decreased phosphorylation of TBK1 and increased cleavage of CASP3, while normal mononuclear cells from healthy donors were relatively insensitive to dinaciclib treatment  expression), or dinaciclib  in AML and represent putative mechanisms of resistance to therapeutically targeting oncogenic NRAS. In the NRAS(V12)-addicted tNM AML mouse model, some mice spontaneously relapsed with NRAS oncogene-independent AML despite continued suppression of NRAS(V12) expression. The spontaneous relapse seen after mimicking RAS oncogene-targeted cancer therapy closely resembles the major clinical challenge for targeted cancer therapies in general,40 and confirm that NRAS(V12)-addicted AML cells can acquire alternative mechanisms for maintained growth and survival and survive in the absence of the NRAS oncogene. With renewed efforts to therapeutically target oncogenic Ras proteins, our results have important implications for anticipating therapeutic challenges for such treatment strategies.42 Specifically, we found that relapsed AMLs developed an NRI mechanism to maintain signaling through RALB. The ability of inhibition of RALB signaling to suppress the clonogenic potential of relapsed NRI AML and to prevent AML relapse in leukemic mice supports further investigation of this pathway as a therapeutic target. Furthermore, the potent antileukemic effect of inhibiting RALB signaling in human AML, including patient-derived AML cells in vitro and in vivo, highlights the translational potential for our findings.In this study, we investigated potential mechanisms that bypass the requirement for 43 Consistent with this, we recently demonstrated a key role for RALB in AML cell survival.37 RALA or RALB activity are essential for cancer cell proliferation in a murine KRAS-driven non-small-cell lung cancer model.44 While RALB has a central role in innate immune signaling, chronically activated RALB associates with SEC5 in the exocyst complex and activates the noncanonical IκB kinase family member TBK1, a critical mediator of RALB’s oncogenic activity.45 The mechanism by which the RALB-TBK1 axis supports cancer cell survival remains unclear and are likely context and disease dependent, but have been shown to involve regulators of normal innate immune signaling in several models.46 A large-scale synthetic lethal RNAi screen uncovered a critical role for TBK1 in KRAS-driven transformation of epithelial cells through activation of nuclear factor-κB antiapoptotic signals involving REL and BCL-xL.38 We found that upregulation of Cdk5 and Rgl2, activators of RALB signaling,35 as well as increased Ralb expression was associated with NRI activation of RALB-TBK1 signaling in relapsed AMLs. Activation of RALB signaling and upregulation of components of the nuclear factor-κB transcriptional complex were accompanied by an altered balance of pro- and antiapoptotic mediators, consistent with previous reports suggesting that nuclear factor-κB signaling downstream of RALB supports cancer cell survival.45 We have previously shown that RALB enhances expression of BCL2 in AML, providing a potential mechanism by which RALB drives NRI AML relapse.37 Consistent with this, BCL2 family inhibitors to suppressed the clonogenic potential of relapsed NRI AML cells; however, the precise mechanisms that promote AML cell survival downstream of RALB remain to be elucidated.There is mounting evidence that the activation and cellular localization of Ral GTPase effector proteins have an important role in Ras-driven transformation, proliferation, migration and survival.47 contributes to the antileukemic activity of dinaciclib. In fact, drugs that target multiple pathways may reduce the ability of AML cells to coopt alternate resistance pathways. Our findings support the further investigation of the critical targets of dinaciclib in AML. New small molecules that directly inhibit Ral function may provide additional tools to study the role and therapeutic potential of RALB signaling in AML as they become available.48While targeting RALB activation with dinaciclib demonstrated potent RALB-dependent antileukemic effects on AML cells, we cannot exclude the contribution of RALB-independent effects. It is possible that dinaciclib-mediated inhibition of CDK1, another protein that was enriched in relapse NRI AMLs, or CDK9, an alternate survival pathway for MLL-rearranged AMLs,RAS oncogene dependence of AML cells. The altered balance of pro- and antiapoptotic mediators in relapsed NRI AML cells that could be a direct or indirect result from enhanced RALB signaling warrants further investigation. YAP, a mediator of the Hippo signaling pathway that has been shown to mediate resistance to inhibition of MAPK signaling and promote Kras oncogene independence in oncogenic pancreatic cancer,25 was enriched in relapsed NRI AMLs. Overall, the broad deregulation of signaling and gene transcription observed provides a foundation for the ongoing rational development of targeted treatment approaches designed to mitigate or prevent relapse after Ras targeted treatment strategies.Our data also identified several other potential mechanisms that may bypass 41 Furthermore, our results support further characterization of RALB signaling as a key mediator of survival and NRAS-independent relapse in AML and as a valid therapeutic target.This work provides important insight into the mechanisms of response and resistance to targeted cancer treatment approaches in general, and particularly for renewed efforts to therapeutically target Ras signaling.P<0.05 using a t-test. For leukemic cell transplantation, 2 × 106 tNM AML cells, 4 × 106 THP1-luciferase AML cells or 2 × 106 patient-derived AML cells were injected via tail vein into 6–10-week-old recipient mice. Severe combined immunodeficiency (SCID) beige  mice did not receive any preconditioning before murine AML transplantation. NRG or NRGS  mice received 375 cGy from an X-ray source 24 h before human AML transplantation. Peripheral blood was obtained by retro-orbital blood sampling and leukocyte counts were monitored using a Hemavet 950 . THP1-luciferase mice were monitored using an IVIS 100 Imaging System . PDX mice were monitored by flow cytometry of peripheral blood stained for human CD45 and human CD33. Doxycycline-treated mice were given 4 mg intraperitoneally doxycycline  followed by 5 mg/ml in their water.All mouse studies were approved by the University of Minnesota Institutional Animal Care and Use Committee. Roughly equal number of male and female mice were used for all experiments and evenly allocated to experimental groups without systematic randomization or blinding. Group sizes were determined based on the number of mice required for 80% power to detect a difference in the mean AML burden of at least 1.5 s.d. between experimental and control groups with in vitro studies inhibitors were diluted in the growth medium. For in vivo studies dinaciclib was diluted in 20% hydroxypropyl-β-cyclodextrin and administered intraperitoneally.Dinaciclib, ABT-737 and ABT-199 were purchased from Selleck Chemicals  and reconstituted in dimethyl sulfoxide (DMSO). For NRD and NRI AML cells were cultured in RPMI-1640  with 10% fetal bovine serum  with or without 1 μg/ml doxycycline (Sigma-Aldrich) for 48 h or inhibitors for 24 h and then plated in IMDM (Lonza) with 30% fetal bovine serum, 1.275% methylcellulose  and 2 ng/ml murine GM-CSF (R&D Systems). Human AML cells were treated with inhibitors for 24 h and then plated in MethoCult H4034 . Colonies were scored after 7–14 days on an inverted microscope.Protein lysates were run on 10% polyacrylamide gel electrophoresis gels and transferred to a PVDF using the NuPAGE and iBlot Systems . RALB-GTP levels were determined using the RALB Activation Assay Kit . Blots were blocked and stained according to antibody manufacturer’s recommendations. Blots were developed using the SuperSignal West Pico ECL  or Advansta Quantum ECL Kit  and signals were quantified using the LI-COR Imaging System .49 Briefly, for intracellular antigens cells were fixed with 2% paraformaldehyde  and permeabilized with 90% methanol (Sigma-Aldrich). Cells were stained with antibodies according to the manufacturer’s recommendations. Cells were analyzed on an LSR II or Fortessa Digital Flow Cytometer  and analyzed using the FlowJo software .Flow cytometric analysis was performed as described previously.For western blotting, NRAS (F155) mouse monoclonal IgG1 (sc-31) and HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology ; BCL-xL rabbit polyclonal (2762) and GAPDH (14C10) rabbit monoclonal (2118) were purchased from Cell Signaling Technologies ; and BCL2 mouse monoclonal (610538) was purchased from BD Biosciences. For flow cytometry, c-MYC (9E10) Alexa Fluor 700, BAD (Y208), BAX (E63), BCL2 (E17), BCL-xL (E18) and BIM (Y36), and MCL1 (Y37) were purchased from Abcam/Epitomics ; phospho-4EBP1 (Thr46) and phospho-mTOR (Ser2448) were purchased from Thermo Fisher; cleaved caspase-3  phycoerythrin, cleaved PARP  Alexa Fluor 647, phospho-4E-BP1  Alexa Fluor 647, phospho-AKT  Alexa Fluor 488, phospho-p44/p42 MAPK  phycoerythrin, BCL-xL (54H6), BIM (C34C5), IkBa (L35A5), PTEN (138G6), phospho-JNK , phospho-S6 (Ser235/236) and phospho-TBK1  phycoerythrin were purchased from Cell Signaling Technologies; PUMA (RB1353-RB1354) was purchased from Novus Biologicals ; and phospho-TBK1  Alexa Fluor 488, cleaved PARP (Asp214), Ki67 (B56), phospho-AKT , phospho-STAT5 , human CD45 (2D1) FITC and human CD33 (P67.6) phycoerythrin were purchased from BD Biosciences.q-value of ⩽0.05.Total splenocytes were harvested from leukemic mice, snap frozen in liquid nitrogen and sent to the MD Anderson RPPA Core Facility for analysis. Briefly, protein lysates were serially diluted and arrayed on nitrocellulose-coated slides using an Aushon 2470 Arrayer . The slides were scanned, analyzed and quantified using Array-Pro Analyzer . Protein concentrations were normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample. Normalized protein values were used to evaluate differential target levels between NRD and NRI samples using a Benjamini–Hochberg-corrected 50 Differential expression between NRD and NRI samples was determined using Cuffdiff.51 R (R core Team 2013) was used to visualize expression data. Genes with RNAseq expression variation over 0.2 were log transformed and mean centered. The resulting data was clustered using Pvclust package with correlation as distance metric and average clustering method.52 Differential transcript expression was defined using a Benjamini–Hochberg-corrected q-value of ⩽0.01. The data discussed have been deposited in NCBI's Gene Expression Omnibus (GSE87870).Total splenocytes were harvested from leukemic mice and RNA was extracted using the RNeasy Midi Kit . RNA samples were quantified, quality checked and analyzed using the Illumina HiSeq 2000  at the University of Minnesota Genomics Core Facility. Raw data was mapped to the mouse mm9 genome using TOPHAT2 suite.m 2-mercaptoethanol (Sigma-Aldrich), 1% GlutaMAX (Thermo Fisher) and 10 ng/ml each of stem cell factor, interleukin-3, interleukin-6, FLT3 (FMS-related tyrosine kinase 3) ligand and thrombopoietin .Cell lines were originally obtained from ATCC or DSMZ, maintained under standard cell culture conditions, tested monthly for mycoplasma contamination and authenticated by STR analysis at the University of Arizona Genomics Core. De-identified mobilized peripheral blood and AML patient samples were obtained after informed consent according to protocols approved by the University of Minnesota Institutional Review Board. Primary AML samples were cultured in IMDM (Thermo Fisher) supplemented with 20% fetal bovine serum , 100 μViable cell numbers were determined using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay . Half-maximal inhibitory concentrations were calculated using CalcuSyn 2.0 .53 by replacing eGFP with eBFP2 and luciferase with RALB(Q72L) (from Channing Der via Addgene plasmid no. 19721), myrAKT (from John Ohlfest) orCRAF W22 (from Channing Der via Addgene plasmid no. 12593). Vesicular stomatitis virus-G-pseudotyped lentivirus was produced by co-transfecting a 1:2:3 ratio of pMD2.G, pCMVDR8.2 (both from Dider Trono via Addgene nos 12259 and 8455) and lentiviral expression vector into HEK293 cells using X-treme Gene HP . Viral supernatant was harvested after 48 h, filtered and used for transduction. Target cells were transduced by coculture with viral supernatant and 5 μg/ml polybrene overnight.Lentiviral expression vectors were generated using the Gateway cloning system (Thermo Fisher). BFP-expressing lentiviral vectors were generated from a previously described backbone,t-test corrected for multiple comparisons using the Holm–Sidak method with PRISM software . Statistical significance was defined as P⩽0.05.Unless otherwise indicated, statistical differences between two groups were determined using an unpaired, two-tailed


Here, we report that patient-derived fibroblasts from three monogenic models of ASD—fragile X and tuberous sclerosis TSC1 and TSC2 syndromes—display depressed Ca2+ release through inositol trisphosphate receptors (IP3Rs). This was apparent in Ca2+ signals evoked by G protein-coupled receptors and by photoreleased IP3 at the levels of both global and local elementary Ca2+ events, suggesting fundamental defects in IP3R channel activity in ASD. Given the ubiquitous involvement of IP3R-mediated Ca2+ signaling in neuronal excitability, synaptic plasticity, gene expression and neurodevelopment, we propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect. These findings highlight potential pharmaceutical targets, and identify Ca2+ screening in skin fibroblasts as a promising technique for early detection of individuals susceptible to ASD.Autism spectrum disorder (ASD) affects 2% of children, and is characterized by impaired social and communication skills together with repetitive, stereotypic behavior. The pathophysiology of ASD is complex due to genetic and environmental heterogeneity, complicating the development of therapies and making diagnosis challenging. Growing genetic evidence supports a role of disrupted Ca Diseases of the intracellular organelles are an emerging area of medicine. Several prototypes are already well-developed for neurogenetic diseases of mitochondria and the lysosomes,14 and increasing evidence implicates the endoplasmic reticulum (ER).15 Ca2+ release from the ER through inositol trisphosphate receptors (IP3Rs) has been shown to be altered in cognitive disorders including Alzheimer's17 and Huntington's diseases,18 and IP3Rs have recently been identified among the genes affected by rare de novo copy number variants in ASD patients.19Cytosolic Ca3R-mediated Ca2+ release is involved in crucial functions—including synaptic plasticity and memory,21 neuronal excitability,23 neurotransmitter release,25 axon growth26 and long-term changes in gene expression27—highlighting the central integrating position played by IP3Rs.28 Ca2+ release is activated in response to the second messenger IP3, which is produced on stimulation of G protein-coupled receptors (GPCRs)29 and tyrosine kinase-linked30 cell surface receptors. The specificity of the resulting cellular responses is ensured by an exquisite temporo-spatial patterning of cytosolic Ca2+ signals.32 Opening of the IP3R channel requires not only IP3, but also binding of Ca2+ to receptor sites on the cytosolic face. This leads to biphasic regulation, such that small elevations of cytosolic Ca2+ induce channel opening, whereas larger elevations cause inactivation.33 The positive feedback by Ca2+ , may remain restricted to individual or clustered IP3Rs, producing local Ca2+ signals known, respectively, as Ca2+ blips and puffs,34 or may propagate throughout the cell as a saltatory wave by successive cycles of Ca2+ diffusion and CICR. Thus, IP3-mediated Ca2+ signaling represents a hierarchy of Ca2+ events of differing magnitudes.36 The spatial patterning it orchestrates is critical to proper cellular function, and we hypothesize that disruptions in the magnitude and organization of neuronal Ca2+ signals may contribute to the pathogenesis of ASD.In neurons, IP37 has been greatly advanced by studies of syndromic forms of ASD caused by rare single gene mutations. Fragile X (FXS) is the most common monogenic cause of ASD,38 and is a widely used and well-characterized model of ASD.39 It results from silencing of the fragile X mental retardation (FMR1) gene and absence of its corresponding protein, the FXS mental retardation protein (FMRP). Tuberous sclerosis (TS) is a syndrome caused by dominant mutations in one of two genes, hamartin (TSC1) or tuberin (TSC2), causing ASD-like behaviors, seizures, intellectual disability and characteristic brain and skin lesions.Our understanding of the etiology of ASD 3-mediated Ca2+ signaling. The physiology of IP3 signaling in fibroblasts has been extensively characterized,42 providing a validated and convenient model for the study of Ca2+ signaling in ASD, with the further advantage that cell lines are readily obtained as clinical samples from both disease and matched control patient populations. Moreover, identification of disease-specific signaling defects in skin cells have potential as biomarkers for diagnostic purposes, much as is now routine in other organelle diseases, such as Tay–Sachs and Niemann–Pick diseases,44 and through which novel therapies for these diseases have emerged.45 Our results demonstrate that IP3-mediated Ca2+ signals are significantly depressed in fibroblasts from both FXS and TS patients and, by resolving signals at the single-channel level, we provide evidence of fundamental defects in IP3R channel activity in ASD. We thus propose dysregulated IP3R signaling as a nexus where genes altered in ASD converge to exert their deleterious effect.Here, we used primary, untransformed skin fibroblasts derived from patients with FXS and TS to evaluate ASD-associated functional deficits in IP3 analog ci-IP3/PM benzyl-myo-Inositol 1,4,5-trisphosphate-Hexakis (propionoxymethyl) Ester) was obtained from SiChem , diluted in 20% pluronic F-127 solution in dimethylsulfoxide to a stock concentration of 200 μM and was frozen down into 2-μl aliquots until needed. EGTA-AM and pluronic F-127 were from Molecular Probes/Invitrogen . Fluo-8 AM and Cal520 were purchased from AAT Bioquest .The membrane-permeant caged IP2, and used for up to 15 passages. Cells were harvested in Ca2+, Mg2+-free 0.25% trypsin-EDTA  and sub-cultured for 2 days before use.Primary, untransformed human skin fibroblasts were purchased from Coriell Cell Repository . ASD cell lines and matched controls with their corresponding Coriell numbers are as follows: FXS-1 (GM05848)/Ctr-1 (GM00498), FXS-2 (GM09497)/Ctr-2 (GM02912), FXS-3 (GM05185)/Ctr-3 (GM03440), FXS-4 (GM04026)/Ctr-4 (GM02185), FXS-5 (GM05131)/Ctr-5 (GM05659), TS1-A (GM06148)/Ctr-6 (GM01863), TS1-B (GM06149)/Ctr-3 (GM03440) and TS2 (GM06121)/Ctr-2 (GM02912). All cell lines came from male Caucasian patients. Cells were cultured in Dulbecco's Modified Eagle's Media  supplemented with 10% (v/v) fetal bovine serum and 1 × antibiotic mix (penicillin/streptomycin) at 37 °C in a humidified incubator gassed with 95% air and 5% CO4 cells per well and grown to confluency. On the day of the experiment, cells were loaded by incubation with 2 μM of the membrane-permeant Ca2+ indicator Fluo-8 AM46 in standard buffer solution  with 0.1% fetal bovine serum for 1 h at 37 °C, then washed with a standard buffer solution. Ca2+-free solution  was added to each well (100 μl), and cells were allowed to equilibrate for 5 min prior to assay with a Fluorometric Imaging Plate Reader . A basal read of fluorescence in each well  was read for 2 s. Next, 100 μl of 2 × ATP  or 100 μl of 2 × ionomycin  in Ca2+-free HBSS was added to each well. Only a single recording was obtained from a given well. Ionomycin-induced fluorescence changes from wells without prior addition of ATP were used to normalize ATP-evoked responses. Recordings were performed in triplicate.Skin fibroblasts were seeded in clear-bottom black 96-well plates  at 1.3 × 103 (ci-IP3).48 Briefly, cells were incubated at room temperature in HEPES-buffered saline  containing 1 μM ci-IP3/PM for 45 min, after which 4 μM Fluo-8 AM was added to the loading solution for further 45 min before washing three times with the saline solution. [Ca2+]i changes were imaged using a Nikon Eclipse microscope system  with a × 40  oil objective. Fluo-8 fluorescence was excited by 488-nm laser light, and emitted fluorescence (λ>510 nm) was imaged at 30 frames per s using an electron-multiplied CCD Camera iXon DU897 . A single flash of ultraviolet (UV) light (350–400 nm) from an arc lamp focused to uniformly illuminate a region slightly larger than the imaging field was used to uncage i-IP3, a metabolically stable isopropylidene analog of IP3, which evoked activity persisting for a few minutes. Image data were acquired as stack.nd2 files using Nikon Elements for offline analysis. Fluorescence signals are expressed as a ratio (ΔF/F0) of changes in fluorescence (ΔF) relative to the mean resting fluorescence at the same region before stimulation (F0). Recordings were performed in triplicate, and the measurement outcomes were compared using Mann–Whitney test.Cells seeded in glass-bottomed dishes were loaded for imaging using membrane-permeant esters of Fluo-8 and caged i-IP2+ signals, cells were incubated at room temperature in HEPES buffer containing 1 μM ci-IP3/PM and 4 μM Cal520 for 1 h,48 washed and further incubated with 10 μM EGTA-AM for an another hour. Cells were then washed three times and remained in buffer for 30 min to allow for de-esterification of loaded reagents. [Ca2+]i signals were imaged using the Nikon Eclipse microscope system described above, but now utilizing an Apo total internal reflection fluorescence × 100  oil objective. The imaging region on the camera sensor was cropped to 128x512 pixels (20.48 × 81.92 μm) to enable rapid (129 frames per s) imaging. Cal520 fluorescence (λ> 510 nm) was excited by 488-nm laser light within an evanescent field extending a few hundred nanometers into the cells. Image acquisition and processing was as described above for whole-cell imaging, except that local events were identified and analyzed using a custom-written algorithm based on MatLab.48For experiments studying local Ca49 About 20 μg of protein was loaded per well with 5% β-mercaptoethanol on 3–8% gradient Tris-Acetate gels with Tris-Acetate SDS running buffer (Invitrogen) and separated by electrophoresis at 130 V. Proteins were transferred at 50 mA for 6 h to 0.2 μm nitrocellulose membranes, which were blocked in 5% nonfat milk in tris-buffered saline supplemented with 0.1% tween-20 for 1 h. Membranes were probed overnight at +4 °C with the following primary antibodies: rabbit polyclonal anti-IP3R1 , rabbit polyclonal anti-IP3R2 , mouse monoclonal anti-IP3R3 , rabbit polyclonal anti-IP3R1/2/3 , rabbit polyclonal anti-beta actin . Membranes were then incubated, as appropriate, with goat anti-rabbit  or goat anti-mouse  HRP-conjugated secondary antibodies for 1 h. Bands were visualized by an ImageQuant LAS 4000 imager  using peroxidase substrate for enhanced chemiluminescence . Levels of protein expression were quantified via densitometry analysis using ImageJ (http://imagej.nih.gov/ij/docs/faqs.html#cite), and are expressed normalized to actin levels.Cell lines were grown in triplicates and lysed in mammalian protein extraction reagent  with complete mini protease inhibitor cocktail tablets  and phosphatase 2 inhibitor cocktail . Lysates were subsequently centrifuged at 14 000 r.p.m. for 15 min at +4 °C. Protein levels in the cell lysate were measured using the Bradford method.3-mediated signaling associated with ASD, we used a FLIPR to monitor cytosolic Ca2+ changes in fibroblasts loaded with the Ca2+-sensitive fluorescent indicator Fluo-8. Primary skin fibroblasts derived from five FXS males and five ethnicity- and age-matched unaffected male donors were grown to confluency on 96-well plates. Cells were stimulated by application of ATP to activate purinergic P2Y receptors51 and thereby evoke GPCR-mediated intracellular Ca2+ release through IP3Rs. Recordings were made in Ca2+-free extracellular solution to exclude complication from Ca2+ influx through plasmalemmal channels. Different concentrations of ATP were applied to individual wells containing FXS and matched control cells. 2+ signals in FXS cells. To determine whether differences in ATP-evoked signals may result from differences in filling of ER Ca2+ stores, we recorded signals evoked in separate wells by application of 1 μM ionomycin in Ca2+-free medium to completely liberate all intracellular Ca2+ stores  or tuberin (TSC2). 2+ signals as compared with matched controls , together with three matched control lines , using antibodies specific to type 1, 2 and 3 IP3Rs, as well as a non type-specific antibody .47 UV flash photolysis of ci-IP3 to photorelease physiologically active i-IP3 then allowed us to directly evoke Ca2+ liberation through IP3Rs in a graded manner by regulating flash duration and intensity to control the amount of i-IP3 that was photoreleased.To then discriminate whether the observed deficits in ATP-induced Ca3 by incubation with membrane-permeant esters of these compounds. F/F0) traces measured from several representative FXS-2 and matched control Ctr-2 cells in response to uniform photolysis flashes. Concordant with our observations of defects in ATP-induced global Ca2+ signals, global cytosolic Ca2+ responses evoked by equivalent photorelease of i-IP3 in these FXS cells were smaller than in control cells . Moreover, we observed a consistent proportional depression of Ca2+ signals for different relative UV flash strengths corresponding to photorelease of different i-IP3 concentrations .ol cells ; and disol cells  and slowol cells . Similar2+ responses to photoreleased i-IP3. Measurements from the matched TS1-B and Ctr-3 cell lines .TS cells also showed depressed and slowed Call lines  revealedplitudes ; and agaplitudes  and the plitudes . These d3-mediated cellular Ca2+ signaling is organized as a hierarchy, wherein global, cell-wide signals, such as those discussed above, arise by recruitment of local, ‘elementary' events involving individual IP3R channels or clusters of small numbers of IP3Rs.52 We therefore imaged these elementary events to elucidate how deficits in the global Ca2+ signals in FXS and TS cells may arise at the level of local IP3R clusters. We selected one FXS (FXS-3) fibroblast line, one TS1 (TS1-B) line and a common control (Ctr-3) cell line matched to both. Ca2+ release from individual sites was resolved utilizing total internal reflection fluorescence microscopy of Cal520 , in conjunction with cytosolic loading of the slow Ca2+ buffer EGTA to inhibit Ca2+ wave propagation.53 This technique captures in real time the duration and magnitude of the underlying Ca2+ flux, providing a close approximation of the channel gating kinetics as would be recorded by electrophysiological patch-clamp recordings.54 Ca2+ release evoked by spatially uniform photolysis of ci-IP3 across the imaging field was apparent as localized fluorescent transients of varying amplitudes, arising at numerous discrete sites, widely distributed across the cell body . Ca2+ responses evoked by agonist stimulation of GPCR-mediated IP3 signaling were significantly smaller in fibroblasts derived from patients with FXS and TS, as compared with matched control cell lines. In contrast, we found no significant differences in Ca2+ liberation evoked by application of the Ca2+ ionophore ionomycin, indicating that the diminished responses to IP3 do not result from diminished ER Ca2+ store content. Moreover, Ca2+ signals evoked by intracellular uncaging of IP3 were depressed in FXS and TS cell lines, pointing to a deficit at the level of Ca2+ liberation through IP3Rs and not solely because of diminished GPCR-mediated production of IP3. Finally, we conclude that the depression of Ca2+ signals cannot be attributed entirely or substantially to reduced expression of IP3R proteins, because mean agonist-evoked Ca2+ responses across four FXS and TS lines were about 22% of matched controls, whereas western blots showed mean IP3R levels to be about 80% of controls and uncorrelated with the extent of Ca2+ signaling depression in these different cell lines.We report abnormalities of IP2+ liberation during ‘elementary', local signals evoked by photoreleased IP3,34 we further demonstrate that defects in global Ca2+ signaling in these three distinct ASD-associated models are reflected at the level of Ca2+ release through individual and small clusters of IP3Rs. In both FXS and TS cell lines, we observed fewer sites of local Ca2+ release as compared with a control cell line, and the durations of these events were shorter. Because functional sites are comprised of clusters of small numbers of individual IP3Rs, the amplitude of the fluorescence signal at a site depends on the channel permeability, together with the number of active channels in the cluster.34 We observed similar amplitudes of local Ca2+ signals across the cell lines, suggesting that the Ca2+-permeation properties and cluster organization of IP3Rs are not appreciably affected in FXS and TS. However, the shorter average duration of local events points to a modulation of IP3R gating kinetics, and would lead to an overall decrease in the amount of Ca2+ released over time. Compounding this, we found the numbers of local Ca2+ release sites within a cell to be dramatically lower in FXS and TS cells as compared with control cells , although it is possible that the short duration events observed in the mutants may have contributed to undercounting their release sites. Taken together, our findings on local IP3-mediated Ca2+ signals indicate that the deleterious effects of FXS and TS mutations manifest at the level of the functional channel gating of IP3Rs, although the underlying molecular mechanism remains to be determined.By resolving Ca3R is a key signaling hub in the canonical metabotropic glutamate receptor (mGluR) pathway in neurons,57 and the mGluR theory of FXS fragile X58 postulates that disrupted mGluR signaling underlies the pathogenesis of the disorder. Activation of mGluRs leads to a brief hyperpolarization followed by a more prolonged depolarization.59 The initial outward current results from the opening of small conductance Ca2+-activated K+ channels.61 This current is proportional to the Ca2+ signal amplitude;23 and can be triggered directly by intracellular uncaging of IP3.59 As a result, IP3-evoked Ca2+ release transiently hyperpolarizes the cell and briefly depresses neuronal excitability, leading to a reduction in firing frequency.23 Suppressed IP3-mediated Ca2+ release from the internal stores, as we report in diverse models of ASD, is thus expected to diminish the inhibitory K+ conductance, and as such would tend to produce neuronal hyperexcitability, consistent with observations following mGluR stimulation of ASD-model neurons.63 A complex array of downstream signals arises from mGluR activation,64 whereas IP3R Ca2+ signaling is one immediate downstream target; to our knowledge its function has not yet been molecularly dissected in ASD. At present, we cannot directly extrapolate our results to IP3-mediated signaling in neurons, given that fibroblasts predominantly express type 3 IP3Rs whereas neurons predominantly express type 1 IP3Rs.65 Nevertheless, because expression levels of all three isotypes of IP3Rs are only slightly diminished in FXS and TS fibroblasts, we conclude that the pronounced depression of Ca2+ signaling does not result from diminished expression of a specific isotype. Instead, the depressed Ca2+ signals likely result from modulatory effects on IP3R function, which might extend across different isotypes.The IP3-mediated Ca2+ signaling may further disrupt neurodevelopment through separate mechanisms. IP3Rs have been shown to be central participants in autophagy.69 Decreased levels of autophagy result in defective synaptic pruning, which has been repeatedly associated with ASD in humans and mouse models,70 and promotion of autophagy rescues behavioral defects in mouse models of ASD.70Depression of IP3R signaling and its diverse roles in almost all cells of the body, deficits in IP3-mediated Ca2+ signaling may not be limited to neurological correlates of ASD, but may also explain other characteristic ASD-associated heterogeneous symptoms, such as those of the gastrointestinal tract72 and immune system.74 Furthermore, since the ER serves as a sensor of a host of environmental stressors, this same mechanism may contribute to the known environmental component to the ASD phenotype, and holds the potential to reveal relevant stressors.Because of the ubiquitous nature of IP3R signaling is affected in three distinct genetic models of ASD, pointing to the ER as a functional ‘hub' where different cellular signaling pathways merge to contribute to the pathogenesis of ASD. In addition to its role in Ca2+ homeostasis, the ER serves as a key integrator of environmental stressors with metabolism and gene expression, as it mediates a host of broad ranging cell stress responses such as the heat shock and unfolded protein responses.75 In this light it can be seen to integrate a matrix of ASD-associated risk factors. We identify the IP3R as a functional target in monogenic models of ASD, and we are currently exploring potential defects in IP3-mediated Ca2+ signaling in ‘typical' ASD patients without any identifiable underlying genetic cause. Ca2+ screening in skin fibroblasts, which are routinely acquired as clinical specimens, may thus offer a promising technique in conjunction with behavioral testing for early detection of ASD, and potentially for high-throughput screening of novel therapeutic agents.In conclusion, our findings indicate that ER IP


Although small changes in arterial CO2 are particularly potent to change CBF , the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E2 (PgE2) plays a key role for cerebrovascular CO2 reactivity, and that preserved synthesis of glutathione is essential for the full development of this response. We combined two-photon imaging microscopy in brain slices with in vivo work in rats and C57BL/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2) evokes an increase in astrocyte [Ca2+]i and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2  depends critically for its vasodilator activity on the level of glutathione in the brain. We show that, when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2 is decreased and vasodilation triggered by increased astrocyte [Ca2+]iin vitro and by hypercapnia in vivo is inhibited. Astrocyte synthetic pathways, dependent on glutathione, are involved in cerebrovascular reactivity to CO2. Reductions in glutathione levels in aging, stroke, or schizophrenia could lead to dysfunctional regulation of CBF and subsequent neuronal damage.Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial COSIGNIFICANCE STATEMENT Neuronal activity leads to the generation of CO2, which has previously been shown to evoke cerebral blood flow (CBF) increases via the release of the vasodilator PgE2. We demonstrate that hypercapnia (increased CO2) evokes increases in astrocyte calcium signaling, which in turn stimulates COX-1 activity and generates downstream PgE2 production. We demonstrate that astrocyte calcium-evoked production of the vasodilator PgE2 is critically dependent on brain levels of the antioxidant glutathione. These data suggest a novel role for astrocytes in the regulation of CO2-evoked CBF responses. Furthermore, these results suggest that depleted glutathione levels, which occur in aging and stroke, will give rise to dysfunctional CBF regulation and may result in subsequent neuronal damage. B1 and B0 are the corresponding background fluorescence signals, taken from the neuropil. Pseudo-color images show absolute changes in fluorescence . Experimental values are mean ± SEM; n is the number of experiments conducted or, for calcium changes, number of astrocytes analyzed. Either a two-tailed Student's t test or a one-way ANOVA with a Newman–Keuls post hoc test for comparison between multiple groups was used, and p < 0.05 was considered statistically significant. As these were novel experiments, the effect size was unknown before the experiment. Therefore, sample size estimates were based on our previous experience. Experiments were alternately performed under control or treatment conditions with slices chosen at random for each experiment. Data were excluded from analysis if any of the following occurred during imaging: unstable baseline vessel diameters or astrocyte calcium levels, or movement leading to significant focus changes during the experiment. To perform statistical analysis, data were assumed to be normally distributed.An image (512 × 512 pixels) was collected in 7.86–12.68 s, using 8-line averaging. Measurements of lumen diameter and Caescribed , fluoresn is the number of animals. To perform statistical analysis, data were assumed to be normally distributed. An F test was used to compare variances of groups being statistically compared. For CBF data, a one-tailed t test with Welch's correction (as groups had significantly different variances) was used to compare means between groups. A two-tailed t test was used to compare means of groups for both GSH analysis (see C) and electrophysiology data in response to whisker pad stimulation . For electrophysiology data collected during hypercapnia challenge experiments, a two-way ANOVA with Bonferroni correction for multiple comparisons was used to compare means between groups. p < 0.05 was considered statistically significant. For experiments involving rats, due to effect sizes being unknown before experiment, sample size estimates were based on previously published sample sizes . To obtain the region of interest (ROI) for calculation of CBF changes using laser speckle imaging, a principal components analysis was used to identify the focal point of the change in response to stimulation. The same region of interest was used within each animal's data. Experimental values are the mean ± SEM, and ysis see C and elees e.g., . Assignmin vivo calcium imaging, frame size was 256 × 256 pixels (189–207 ms/frame) during recordings. The Ca2+ changes were evaluated as the average change in fluorescence relative to baseline levels in ROIs. The ROIs were placed based on morphology over neuronal or astrocytic soma, or neuropil. Because of movement of astrocytes during hypercapnia, within or out of focus, ROIs were evaluated based on the level of SR101 loading in the red channel. If a significant change occurred, the ROI was disregarded in all following assessments. An increase in fluorescence within an ROI was classified as a calcium response if the mean fluorescence value within the period of hypercapnia was >2 SDs of baseline activity. The delay of the Ca2+ response was found by subtracting the signal onset time from the time hypercapnia was introduced to the animal. To estimate response start and termination time, a fit was made to the data and the first- and second-order derivatives were calculated. The response onset time was found by taking the maximum peak of the second order derivative of the fitted data. The duration of the Ca2+ response was then found by subtracting the response onset time from the response termination time. The response termination time was defined as the time point when the fitted data went below mean baseline Ca2+ levels or the recording ended. Experimental values are expressed as mean ± SEM. A paired t test was used for the calcium imaging data, each animal served as its own control. p < 0.05 was accepted as statistically significant. For experiments involving mice, as there have been no previous studies reporting astroglial calcium changes during hypercapnia, it was impossible to estimate an expected value for change in fluorescence or its SD. Hence, no sample size calculation could be performed. However, we expected similar calcium changes to those we observe for low-frequency whisker stimulation, and so sample sizes were based on our previous experiments (6–8 mice). Calcium signals obtained during hypercapnia exceeded an SNR of 4:1, and hypercapnia-induced calcium responses were recorded in every animal tested. As all mice were subjected to hypercapnia, there was no randomization method used. Control measurements of calcium activity  were taken at random time points during the experiment. Analysis of calcium changes was not blinded, assessment of these changes was based on a MATLAB program, which analyzes the image sequences in an unbiased manner, rather than by visual inspection.For 2 concentration, which can be caused by neuronal metabolism, is known to dilate cerebral blood vessels in a process dependent on PgE2  have not been resolved. Astrocytes can produce PgE2, but it is unknown whether astrocytes generate [Ca2+]i signals in response to CO2. Therefore, we tested whether an increase in inspired CO2 (hypercapnia) in vivo evokes astrocyte [Ca2+]i when it also triggers CBF increases.Elevation of tissue CO on PgE2  formatio on PgE2 . Howeverin vivo was used to examine the simultaneous responses of both neurons and astrocytes to hypercapnia in the intact brain as a first step to investigate which cell type might be the primary sensor of CO2  than increases in [Ca2+]i observed in neuronal soma during the period of hypercapnia. The number of astrocytes with [Ca2+]i responses was also much greater in hypercapnia compared with the number showing spontaneous calcium activity . Although neurons could display increased [Ca2+]i during hypercapnia, with onset times within seconds , there was no significant difference in the number of neurons with [Ca2+]i responses during hypercapnia compared with the number showing spontaneous calcium activity . Measurements taken in the neuropil where there were no defined cell bodies, and it is difficult to separate signals in fine astrocyte processes from neuronal processes did not show correlated changes in [Ca2+]i signals during hypercapnia . The astrocyte [Ca2+]i responses  appear to occur within a similar timescale as the increased CBF evoked by hypercapnia . During hypercapnia, an increased number of astrocyte soma  displayed increased [Ca2+]i with onsets within seconds  and variable durations of tens of seconds . While there were no differences between the three groups  with regards to the delay of the hypercapnia-induced Ca2+ responses , neuropil = 12.83 ± 4.18 s (n = 3), and astrocyte soma = 14.57 ± 1.55 s (n = 47)), the average Ca2+ response duration  was found to be significantly longer in astrocytes than in neurons: neuron soma = 119.41 ± 8.82 s (n = 33), astrocyte soma = 155.47 ± 8.32 s (n = 47) .Two-photon laser scanning microscopy (2PLSM) r of CO2 . Remarkaof mouse  during tin vivo that hypercapnia evokes an increase in astrocyte [Ca2+]i, we then used a combination of 2PLSM and PgE2 measurements using ELISA in acute brain slices to determine the mechanistic links between astrocyte [Ca2+]i responses and CBF regulation. Using a biochemical model, we investigated the role of GSH in the generation of PgE2.Having demonstrated in vivo situation, it is difficult to reliably evoke astrocyte [Ca2+]i signals and vasodilations by applying CO2 to acute brain slices. Thus, we needed an alternative method of elevating astrocyte [Ca2+]i in acute brain slices. Although the adult mouse  to brain slices (from juvenile rats) caused a generalized increase in astrocyte [Ca2+]i, observed using 2PLSM (A–C), that provided us with the ability to measure subsequent synthesis of PgE2. Applying tACPD resulted in the formation and efflux of PgE2, as measured by ELISA (D). The first step in the conversion of AA to PgE2 in astrocytes is via COX-1  in the presence of the COX-1 inhibitor SC560 . Thus, astrocyte COX-1 activity is required for the subsequent PgE2 release in acute brain slices, which is triggered by astrocyte [Ca2+]i signals.Unlike in the lt mouse  and rat lt mouse  have bee animals . Therefong 2PLSM A–C, thatby ELISA D. The fiia COX-1   (B). Therefore, we investigated whether PgE2 formation was reduced when GSH levels were depressed. We examined whether there is a reduction in astrocyte [Ca2+]i-evoked PgE2 release in hippocampal slices after treatment with BSO (an inhibitor of γ-glutamylcysteine synthetase) for 2.5 h . When GSH levels were decreased, although there was no change in basal PgE2 efflux  or in the amplitude of tACPD-evoked astrocyte [Ca2+]i signals , strikingly the tACPD-evoked PgE2 efflux was reduced by 64% .Downstream of COX-1 the synthesis of PgE mPgES-1 , a form  mPgES-1 A (TachikES-1 (A)  that reqES-1 (A) . It is kES-1 (A) , as deteES-1 (A) B. Therefor 2.5 h , which r2 release , which was abolished in the presence of SC560 , whereas the amplitude of evoked astrocyte [Ca2+]i signals was unchanged . Thus, combined with the results discussed above, these data confirm that astrocyte COX-1 activity and subsequent PgE2 release are required for vasodilations in acute brain slices that are triggered by astrocyte [Ca2+]i signals.Bath perfusion of tACPD induced arteriolar dilation in acute brain slices A,F,G, wh2 involves the astrocyte-expressed, GSH-dependent, enzyme mPgES-1 . However, the vasodilations triggered by these [Ca2+]i signals were abolished . Vasoconstrictions evoked by NE (100 μm) or the α2 agonist clonidine (10 μm), which act directly on arteriole smooth muscle cells , indicating that arterioles were not damaged by the BSO treatment. Furthermore, BSO treatment did not alter the vasodilation evoked by either 1 μm PgE2  or high [K+] (10 mm), which causes vasodilation by hyperpolarizing arteriole smooth muscle cells .As previously discussed, downstream of COX-1, the synthesis of PgE mPgES-1 . Therefonaltered A–C. Howele cells , were unle cells   and elicited vasodilation of the neighboring arteriole . Although astrocyte [Ca2+]i signals were unaltered following BSO treatment to reduce GSH levels , dilations were not observed and vasoconstrictions were now evoked . Thus, when GSH levels are reduced, astrocyte [Ca2+]i signals can no longer evoke vasodilations normally triggered by the release of PgE2.Astrocyte [Ca2+]i-evoked vasodilations, we examined whether these same enzymes and molecules were involved in the CBF response, which occurs downstream of CO2-evoked astrocyte [Ca2+]i responses in vivo. Hypercapnia in vivo evoked a CBF increase in the barrel cortex of adult rat , whereas neural activity was unchanged (F). The calculated area under the curve (AUC) of the CBF response was significantly attenuated by SC560 , confirming that COX-1 plays a critical role in hypercapnia-evoked CBF increases in vivo . Treatment with BSO reduced the hypercapnia-evoked CBF response . Neural activity was no different in BSO-treated rats compared with saline-treated rats . Combining all the data described so far suggests that hypercapnia-evoked, astrocyte [Ca2+]i-related, CBF increases require PgE2 release and, thus, are compromised when brain GSH levels are reduced.We examined the impact of decreased tissue GSH levels on COin vivo on functional hyperemia in the somatosensory cortex. Whisker pad stimulation (10 Hz) evoked a blood flow increase in the barrel cortex (A). In agreement with previous findings  or evoked neural activity (LFP) . Furthermore, the AUC of the stimulation-evoked CBF response was not significantly different in BSO-treated animals  compared with saline-treated animals, demonstrating that the CBF response is not GSH-sensitive. The magnitude of the neural response to whisker pad stimulation was unaffected by BSO . These results indicate that, under these experimental conditions, COX-1 and GSH play little, if any, role in the CBF response to somatosensory stimulation. These findings confirm that several different pathways exist that account for CBF regulation under differing conditions and in response to different stimuli.This finding was specific to hypercapnia-evoked CBF increases. We examined the impact of decreased tissue GSH levels l cortex A. In agrfindings , inhibitin vivo that, upstream of evoked COX-1 activity, CO2 increases [Ca2+]i in astrocytes. These data demonstrate a new signal (hypercapnia) that activates astrocyte calcium and specifically identify the involvement of astrocytes in the regulation of CBF in response to changes in arterial CO2.We demonstrate a novel mechanism of CBF regulation involving astrocytes, which is GSH dependent. Previously, In vitro, using brain slices from juvenile animals in which it is possible to examine calcium signals by bulk loading a calcium indicator dye, we confirm that increased astrocyte [Ca2+]i results in the subsequent release of PgE2 and vasodilation which are COX-1 activity-dependent  (A) (B)   , mPgES-1 (COX-1) A (TachikX-1) (A) , and GSHX-1) (A) B (Sun et (A) (B) , providi (A) (B) , is the cofactor . In vitrs of GSH B (Sun et GSH (B) , the depdilation .2+]i signals, under certain conditions and not under others.This GSH-dependent mechanism of CBF regulation exists alongside other COX-1 and GSH-insensitive mechanisms. For example, we found no effect of blocking COX-1 activity or of lowering GSH levels on CBF responses following 10 Hz whisker pad stimulation. Although it is possible that an astrocyte calcium response  may be evoked by an intense sensory stimulus , our res2+]i, increases and changes in CO2 concentration. For example, within the respiratory center, increased astrocyte [Ca2+]i, and astrocytic release of ATP can be triggered by CO2-evoked decreases in pH  (2 (which contributes to neurovascular coupling)  , are capoupling) . In thisfindings . Future findings  to confi2+]i signals, increase CBF in response to CO2. Astrocytes are therefore poised to detect the metabolic activity of neurons and to modify vascular tone appropriately to deliver glucose and O2. This important pathway may be impaired in conditions in which oxidative stress reduces GSH levels in astrocytes, leading to impaired CBF responses and altered vascular readouts of neural activity.In conclusion, we demonstrate a novel mechanism by which astrocytes detect hypercapnia and, via [Ca


Ca2+ release, Ca2+ decay and sarcomere deformation. Local deformation heterogeneities can occur due to a combination of factors: regional/local differences in Ca2+ release and/or re-uptake, intra-cellular material properties, sarcomere proteins and distribution of the intracellular organelles. To investigate the possible causes of these heterogeneities, we developed a two-dimensional finite-element electromechanical model of a cardiomyocyte that takes into account the experimentally measured local deformation and cytosolic [Ca2+] to locally define the different variables of the constitutive equations describing the electro/mechanical behaviour of the cell. Then, the model was individualised to three different rat cardiac cells. The local [Ca2+] transients were used to define the [Ca2+]-dependent activation functions. The cell-specific local Young’s moduli were estimated by solving an inverse problem, minimizing the error between the measured and simulated local deformations along the longitudinal axis of the cell. We found that heterogeneities in the deformation during contraction were determined mainly by the local elasticity rather than the local amount of Ca2+, while in the relaxation phase deformation was mainly influenced by Ca2+ re-uptake. Our electromechanical model was able to successfully estimate the local elasticity along the longitudinal direction in three different cells. In conclusion, our proposed model seems to be a good approximation to assess the heterogeneous intracellular mechanical properties to help in the understanding of the underlying mechanisms of cardiomyocyte dysfunction.Experimental studies on isolated cardiomyocytes from different animal species and human hearts have demonstrated that there are regional differences in the  Ca2+  decay time, indicating that dyssynchrony of the decay of cytosolic [Ca2+] contributes to dyssynchronous intracellular sarcomere re-lengthening. Additionally, some studies have shown that sarcomere relaxation is not uniform in striated muscle [Ca2+ removal [Ca2+] decay and sarcomere re-lenghtening may impair cardiomyocyte function  to locally determine the different parameters of the constitutive equations. Hence, the resulting finite element electromechanical model of a cardiomyocyte accounts for the heterogeneities in the mechanical properties of the cell. We assessed the relationship between cell contraction and regional mechanical properties such as elasticity, and the amount of active force generated by the sarcomeres and cytosolic [Ca2+], in an effort to explain the observed local differences in the amount and duration of cell contraction. Local cytosolic [Ca2+] transients and local deformation along the longitudinal direction of the cell were measured by confocal line-scan microscopy, in three electrically stimulated rat cardiac cells. This data was used to individualise the model and to estimate the local Young modulus along the longitudinal axis of the cell by means of an inverse finite element problem, which minimises the error between the experimentally measured local deformation and the one obtained from numerical simulations.The aim of this study is to develop a simplified two-dimensional electromechanical cell model that takes into account the experimentally measured local deformation and cytosolic  transients (F) were obtained for all points of the line-scan where local strain was manually measured, from the [Ca2+]-dependent fluorescence intensity line-scan images .The local -dependent function modelling the excitation-contraction coupling. Parameters εopt, s and fmax were identified from experimental data acquired on skinned rat cardiac myocytes [log[Ca2+]) ranging from 4.9 to 5.7 with the expression of active stress Tact  depends on the amount of cytosolic Ca2+ available. Therefore, A was defined as an activation function modelling the [Ca2+]-dependent contraction process. This process was described as follows:nH is the Hill coefficient (nH > 0.0), Zmax represents the maximum peak of cytosolic [Ca2+] and Z50 the half-maximum concentration of cytosolic [Ca2+]. The second term, Z(t)/Zmax, corresponds to the normalised [Ca2+] transient. Z(t) was approximated as a sum of two exponential functions as follows:a and b define the amplitude and baseline of the [Ca2+] transient and kfall and krise the rate constants of [Ca2+] decay and rise respectively. Therefore, the corresponding time constants were calculated as τfall = 1/kfall and τrise = 1/krise. For our simulations, the Hill coefficient nH was set to 2.6 following [Z50 was calculated from the global [Ca2+] transient. The last term in fTCa(t), is a unitary function that describes the temporal behaviour of the [Ca2+] binding to and dissociation from troponin (TnC) in the myofilaments, and therefore the [Ca2+] regulation of contraction  heterogeneity, A(t) was defined locally at each point of the line-scan. Local and global [Ca2+] transients were obtained from experimental data. Ca2+] transients respectively and Z(t) fitted to the global [Ca2+] transient. Note that using the actual [Ca2+] transients measured in the cell, makes our simulation independent of the stretch-sensitive channels present in the cell membrane, that will otherwise be important in a fully electro-mechanical analysis of the cell contraction. Ca2+ transients and the dynamic force-Ca2+ phase-plane plots for a single contraction alongside the steady state plot respectively, showing a good agreement with what was experimentally measured in rat left ventricular cells  transient decay , (3) Young’s Modulus (E), (4) fmax, (5) εopt and (6) s. The output variables were: (1) maximum contraction in the longitudinal direction (τsl) and (3) maximum strain rate in the longitudinal direction   transients were defined by randomly varying the amplitude . Then, observations were obtained using the simulated longitudinal strain at maximum contraction time frame, and considered as the experimental strain for the inverse problem. Subsequently, starting from a given parameter set E, different from the one used for the forward simulation, the inverse problem described previously was solved to recover the original (synthetic) local Young’s modulus. The robustness of the method in the presence of imprecise measurements was evaluated by adding % of additive white gaussian noise to the local deformation issued from the forward problem, as done in similar in-silico procedures  transients (F/F0) to The activation function Ca2+]-dependent activation in the whole cell  was assumed. The second scenario considers the different local [Ca2+]-dependent activation functions, calculated from the experimentally measured local [Ca2+] transients.Two possible scenarios were considered. First, a homogeneous   and of heterogeneous (blue line) [Ca2+]—activation. The figure demonstrates the heterogeneity in the longitudinal strain during deformation. In addition, the two simulated strain curves were quite similar compared to the experimentally measured strain. The local estimated Young’s moduli along the line-scan is plotted for both simulated scenarios in The cell-specific electromechanical simulations were successfully performed for three different cardiomyocytes, both with homogeneous and heterogeneous  decay  . The results from the cell-specific simulations also support this hypothesis, since local cell elasticity estimated when considering an homogeneous [Ca2+] activation function was practically identical to the local elasticity estimated when the local [Ca2+] transients were used to construct the activation functions. This suggests that the regional variation of the cell’s elasticity is orders of magnitude higher than changes induced by the [Ca2+] heterogeneity, and therefore a global homogeneous activation could be considered instead. Also, the averaged Young’s moduli identified in our simulations and the cell’s global shortening were in close agreement with reported values for rat cardiac cells  signal was not considered and the variation of Young Modulus through the cell beat (dynamic Young Modulus) neither. Nevertheless, this was out of the scope of the study since we were only interested in the estimation of the static Young’s modulus. The tension dependence Ca2+ binding to TnC was indirectly modelled through the function fTCa(t). In this study, due to the lack of force measurements, it was fitted to the normalised cell deformation to catch the time course of Ca2+ binding to and dissociation from TnC. However, if force measurements were available fTCa(t) can be fitted to the force transient and therefore modelling the dependence Ca2+ binding to TnC. We did not include a detailed model for Ca2+ dynamics and the associated propagating calcium waves. We acknowledge that these kind of models can provide more information about the factors that can lead to heterogeneities in the Ca2+ transient. However, our model considered the local experimentally measured Ca2+ transients instead, which allow us to parametrise the model for each specific cell. Regarding the Ca2+ wave propagation, healthy rat ventricular cells have a very high dense T-Tubule network, thus, in a freely contracting cell, the Ca2+ release is taking place almost at the same time along the cell. This can be appreciated in the line-scan image corresponding to cytosolic [Ca2+]  captured in our active contraction model by the term A , it is believed that a 2D model is accurate enough for reproducing the experimental data. For this reason , only heterogeneity in the longitudinal direction of the cell was considered while homogeneity in the transversal direction was assumed. Secondly, the viscous component of the cardiomyocyte and the effect of velocity on contraction were not modelled, and therefore the dependency on the frequency of the [c [Ca2+] , where ne term A  describira et el  which alIn conclusion, our proposed electromechanical model of a cardiomyocyte seems to be a good alternative to assess the local mechanical properties such as passive elasticity within a normal cardiomyocyte and offers a relevant basis for understanding the spatial heterogeneity in the mechanical properties and deformation. Our results suggested that the heterogeneity in the local deformation is mainly caused by the regional variation on the cell elasticity that could be attributed to local changes in the cytoskeleton of the cell and the different organelles. Therefore, this model could provide a starting point for an extended analysis of the relationship among regional cell mechanical behavior and the underlying biochemical and/or structural environment, in both physiological and pathological contexts.S1 Appendix(PDF)Click here for additional data file.S1 FigTact given by equation S1.22 in Fitting of the experimental data obtained by Weiwad et al , highlig(TIFF)Click here for additional data file.


The aim of mentorship is to build the mentees capacity, enhance their skills and improve their ability to produce desired outcomes. However, the mentoring relationship is vulnerable to a number of challenges that may undermine its effectiveness and sustainability. We aimed to explore the experiences and perceptions of student and junior faculty mentees and senior faculty mentors at the Makerere University College of Health Sciences and identify the key factors defined by mentees and mentors as necessary for a successful mentorship program.A qualitative design involving focus group discussions (FGDs) and key informant interviews (KII) was used. A total of eight KII and four FGDs were conducted, audio recorded and transcribed verbatim. Open coding of the transcripts was performed, and major themes were identified through multiple readings based on thematic analysis.Six key themes were shared by the mentees and mentors including: 1) defining the role of the mentor; 2) desired characteristics of a mentor and a mentoring relationship, with an emphasis on mutual trust and respect; 3) overlapping roles of mentors and supervisors; 4) issues with the process for identifying mentors, including the benefits and drawbacks of the mentee selecting mentor vs. being assigned a mentor; 5) current barriers to mentoring, including lack of knowledge about current program, lack of formal structure, uncertainly about who should initiate relationship, and unclear roles and expectations and 6) recommendations for the future development of mentoring programme, including the need for a formalized programme, and training adapted to the local context.The mentees and mentors described the role of the mentor and desired characteristics of mentors and a mentoring relationship similarly. Most concerns about mentoring occurred when current mentoring programmes and practices were not well aligned with these desired characteristics. Recommendations for future development of mentoring included greater formalization of mentoring with mentoring programmes based on shared expectations and adapted to the local context. In an ideal mentoring relationship, an experienced, highly regarded, empathic person, the mentor, works with and guides another individual, the mentee, in the discovery, development and examination of their own ideas, learning and personal and professional development . It is aMentoring creates opportunities for guidance, knowledge and support in a way that the mentee can willingly benefit from the mentoring relationship .Mentorship in the academic health sciences has an important symbiotic relationship as the mentee is not an empty vessel receiving the mentor’s advice and wisdom but rather, an active participant, shaping the relationship –6. PreviSpecifically, previous research has shown that mentors contribute significantly to the development of their mentees’ research, teaching and clinical skills, particularly with respect to career satisfaction, career management and collegial networking , 5, 7, 9The mentoring relationship however is prone to a number of challenges that may undermine its effectiveness and efficiency. Previous studies have indicated several challenges including: a limited pool of mentors, insufficient understanding of the mentoring process, an excessive workload for the mentor or mentee that precludes regular or in-depth mentorship meetings, and responsibilities and needs that undermine the sustainability of the mentoring programmes, e.g., clinical, research or educational demands on time –12.Since mentoring relationships are an invaluable way of stimulating critical thinking and are key to developing productive careers; there has been increasing and significant interest in mentorship at Makerere University College of Health Sciences (MakCHS) Uganda , 13. A nMentoring in the five clinical departments of MakCHS includes faculty-student mentorship and senior faculty-junior faculty mentorship . FacultyIt is mandatory for every post-graduate student at MakCHS to have a mentor. The mentee is assigned a mentor by their department. Each department handles the process of assigning mentors differently with mentees having no choice of mentor in any of the departments. All faculty members are eligible to be assigned as mentors with departments making decisions about who will be selected as a mentor and for how many mentees. Mentors do not currently receive any systematic training in mentoring through their departments or the college. However, there have been voluntary workshops on mentoring offered periodically. The current mentoring programming is largely unstructured beyond the assigning of mentors and the expectation that there will be one-on-mentoring between mentors and mentees throughout the mentees program of study. The mentoring of junior faculty is less well defined with junior faculty members identifying their own mentors and no formal expectations of the mentoring by the departments or college.Most of the previous studies on mentorship have been conducted in high-income countries (HIC). In fact, all of the studies cited in two recent systematic reviews of mentoring were from HIC , 7. WhilThe previous research on mentoring in LMIC institutions is limited with most previous studies focusing on the mentorship of global health researchers often by HIC mentors with an emphasis on research capacity-building in LMIC –21. StudThe objectives of our study were to explore and obtain a deeper understanding of the experiences and perceptions of mentoring of postgraduate students, junior faculty mentees and senior faculty mentors in the clinical departments at MakCHS specifically related to their perceptions of successful mentoring relationships, challenges and barriers to mentoring that they have experienced and recommendations for improvement. Our goal was to identify key factors needed for a successful mentorship program that would be useful not only to MakCHS but to other medical schools globally and particularly to colleges of health sciences in Africa and other low and middle income countries where institutionalized mentoring programmes have not yet taken hold .We conducted an exploratory qualitative research involving focus group discussions (FGD) with post-graduate students (mentees) and key informant interviews (KII) with faculty members (senior faculty mentors and junior faculty mentees).Mentees and mentors from five departments were invited to voluntarily participate in the study by email and notices placed in the postgraduate study rooms and departmental notice boards. The five departments were purposefully chosen to provide variability in number of mentees (including departments with many and departments with few students) and availability of faculty to participate in the study. We purposively recruited mentees from all 3 years of training and mentors with a range of years of experience in order elicit multiple perspectives on the mentoring programme.We present data of twenty-three postgraduate student mentees including eleven from Internal Medicine, six from Paediatrics and Child Health, and six from Psychiatry. Of the 23 mentees, 6 (26%) were first year students and 17 (74%) were second or third year students. Eight faculty members agreed to participate in the study including two each from Paediatrics and Child Health, Psychiatry and Surgery, one from Obstetrics and Gynecology and one senior administrator from the central administration of MakCHS.All participants were aware of the mentoring programme at MakCHS and had at least some involvement with the programme. In general, the first year mentees had the least experience having been students for 8 months at MakCHS when the study was held. All first year mentees had at least made initial contact with their mentors. The second and third year mentees had more experience in the mentoring programme having had more interaction with their mentors though the amount of interaction varied. For the faculty members, all were current mentors in the mentoring programme. The senior administrator is also a senior faculty member and has been a mentor in the program as well as being involved in the administration of the mentoring programme.A qualitative research design, which is an interpretative method of naturalistic inquiry with a focus on exploring, describing, and interpreting socio - personal experiences and meanings participants attach to their social world represented in language , 23 was Five focus group discussions  and eight key informant interviews were conducted from May – July 2013. We present data from four FGDs as the audio recording from the Obstetrics/Gynecology group was almost inaudible, therefore no accurate transcription could be done. All interviews were conducted in English (the language of instruction at MakCHS) by an experienced interviewer (AS) using a KII schedule and FGD guide  defining the role of the mentor; 2) desired characteristics of a mentor and the mentoring relationship; 3) the overlapping roles of mentor and supervisor; 4) the process for identifying mentors; 5) current barriers to effective mentoring, and 6) recommendations for the future development of mentoring programmes  the design and establishment of a formal mentorship programme for the graduate students and faculty; b) developing mentoring metrics through which the progress of the mentorship programme can be measured; c) longitudinal studies of mentees to determine the longer-term impacts of mentorship. Mentorship is critical, as the post-graduates will become future mentors and essential to the future success of the mentorship programme.


However, continuous Ca2+ patterns during morphogenesis have not yet been studied using fluorescence resonance energy transfer to track the Ca2+ sensor. In the present study, we monitored Ca2+ levels during zebrafish morphogenesis and differentiation with yellow cameleon, YC2.12. Our results show not only clear changes in Ca2+ levels but also continuous Ca2+ patterns at 24 hpf and later periods for the first time. Serial Ca2+dynamics during early pharyngula period  was successfully observed with cameleon, which have not reported anywhere yet. In fact, high Ca2+ level occurred concurrently with hindbrain development in segmentation and pharyngula periods. Ca2+ patterns in the late gastrula through segmentation periods which were obtained with cameleon, were similar to those obtained previously with other Ca2+sensor. Our results suggested that the use of various Ca2+ sensors may lead to novel findings in studies of Ca2+ dynamics. We hope that these results will prove valuable for further research in Ca2+ signaling.Intracellular calcium ion (Ca Ca2+ is relevant to most biological phenomena, and is particularly relevant to early development  requirement of the substrate coelenterazine which is gradually consumed, (2) difficulty of detecting subtle signals which is weaker than the one fluorescent Ca2+ sensor emits, (3) occasionally unsuitable for a long-term and high-speed photography. To present, ‘continuous’ Ca2+ patterns such as long-term time lapse imaging in zebrafish morphogenesis after 24 hpf (hour post fertilization) have not been reported yet. Meanwhile, stable Ca2+ signals are expected with fluorescent Ca2+ sensors such as yellow cameleon YC2.12 because the sensor molecule is integrated into cells. This is advantageous in long-term measuring since Ca2+ sensor is synthesized in vivo and does not require a substrate like luminescent Ca2+ sensor does. Fluorescence emits stronger light than luminescence in general although requiring an excitation light, which enables us to measure real-time and to detect subtle signals.Intracellular calcium ions  to confirm YC2.12 would work correctly (Experiments were conducted as previously described . Brieflyorrectly . The num2+ patterns showed dynamic changes during zebrafish morphogenesis (2+ monitoring had been well studied with aquorin by 2+ levels were observed in the anterior and posterior body regions from stages bud to 16-somite (10–17 hpf). In the anterior trunk, the Ca2+ level reached a peak at 18-somite stage, whereas in the posterior trunk the Ca2+ peak was shown at 28-somite stage , it is quite interesting to consider relevance between Ca2+ signals and formation of neuronal network. Ca2+ involves with neural network in zebrafish and Ca2+ sensors were used for studying neuronal activity and reflexive behavior  that we obtained with yellow cameleon, YC2.12, were similar to those obtained previously with aequorin  which have not reported anywhere yet.In the developing tail, the Caaequorin . However2+ dynamics in the late gastrula-segmentation periods have been well characterized by Webb, Miller and colleagues  Trunk area of zebrafish embryo. (B) CaClick here for additional data file.10.7717/peerj.2894/supp-2Figure S22+ patterns at rhombomere region at 26-somite to prim-10 stages. Scale bar, 200 µm.(A) Developing hindbrain and schematic rhombomeres (r1-7) of zebrafish embryo. (B) CaClick here for additional data file.10.7717/peerj.2894/supp-3Figure S32+ patterns (upper) coincided with CaMK-II gene expression patterns (lower) at (A) 3-somite, (B) 18-somite and (C) prim-5 stages. Schematic images of CaMK-II expressions were created based on CaClick here for additional data file.10.7717/peerj.2894/supp-4Figure S42+ pattern (upper) and bright field image (lower) of the normal embryo. (B) Ca2+ pattern (upper) and bright field image (lower) of the embryo treated with thapsigargin 2.5 µM for 10 m. The control experiment showed that YC2.12 was working correctly as Ca2+ sensor. Scale bar, 200 µm.YC2.12 injected zebrafish embryos were treated with thapsigargin at oblong stage. (A) CaClick here for additional data file.10.7717/peerj.2894/supp-5Data S1Click here for additional data file.


ATP produced a rapid increase in [Ca2+]i consisting of an initial transient elevation followed by a sustained elevated phase that could be observed only in the presence of extracellular Ca2+. Gene expression data and [Ca2+]i recordings with uridine-5′-triphosphate or with the phospholipase C (PLC) inhibitor U73122 demonstrated the involvement of purinergic P2Y2 receptors and the PLC/inositol trisphosphate pathway. The [Ca2+]i elevation produced by reintroduction of a Ca2+-containing intracellular solution to adipocytes exposed to ATP in the absence of Ca2+ was diminished by known SOCE antagonists. The chief molecular components of SOCE, the stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1), were detected at the mRNA and protein level. Moreover, SOCE was largely diminished in cells where STIM1 and/or ORAI1 had been silenced by small interfering (si)RNA. We conclude that extracellular ATP activates SOCE in white adipocytes, an effect predominantly mediated by STIM1 and ORAI1.In the present study, we have applied ratiometric measurements of intracellular Ca White adipose tissue (WAT) is an organ distributed at several locations in the body and with an essential role in the regulation of energy homeostasis. WAT is primarily composed of white adipocytes involved in the modulation of lipid metabolism as well as in the storage of fatty acids. Moreover, adipocytes have an endocrine role and release bioactive substances such as cytokines and hormone-like substances . Adipose2+ concentration ([Ca2+]i) are vital to a wide array of cell biological processes such as signal transduction, muscle contraction, control of enzyme activity and regulation of exocytosis. However, cytoplasmic Ca2+ excess resulting from elevated Ca2+ influx, disturbances of intracellular Ca2+ sequestering and/or malfunctioning plasma membrane Ca2+ ATPases may cause cytotoxicity and trigger cell death. Thus, [Ca2+]i needs to be tightly regulated [2+]i. In electrically excitable cell types, plasma membrane depolarization usually leads to activation of voltage-gated Ca2+ (Cav) channels allowing influx of Ca2+ from the extracellular space [2+ from endoplasmic reticulum (ER) stores [2+ entry  is a major [Ca2+]i elevating pathway present in most eukaryotic cells. In SOCE, depletion of intracellular Ca2+ ER stores leads to activation of plasma membrane-bound store-operated Ca2+ channels (SOCCs) and subsequent influx of Ca2+ from the extracellular space. Although the pathway by which depletion of intracellular calcium stores activates calcium entry was described 30 years ago i show that extracellularly applied ATP (100 µM) stimulates SOCE via interaction with P2Y2 receptors leading to activation of the PLC/IP3 pathway. The SOCE is inhibited by known SOCC antagonists as well as by siRNA  knockdown of STIM1 and/or ORAI1. Moreover, we confirm the presence of STIM1 and ORAI1 at the protein level.Here, we have investigated SOCE and the functional existence of SOCCs in 3T3-L1 adipocytes. Dynamic measurements of i were recorded by dual-wavelength microfluorimetry. Using minimal light, fura-2AM was excited at 340 and 380 nm by the use of a Lambda DG-4 filter system. Emitted light was collected above 510 nm. The data were recorded using MetaFluor software and calculated into absolute concentrations using equation 5 of i was measured as described above.Cells were transfected with either Silencer® or Silencer® Select pre-designed siRNA (Ambion) at differentiation day 6 in Opti-MEM medium using 100 nM siRNA and lipofectamine 2000 (Life Technologies). siRNA identification: 64735 and s74488 [Stim1], s99510 and s99511 [Orai1], s75482 [Trpc1] and negative control #1 siRNA. Medium was replaced 8 h after transfection by antibiotic-free DMEM (10% FBS). The cells were used for experiments 60 h after the start of transfection procedure. Knockdown was validated by qPCR (quantitative polymerase chain reaction), and i imaging experiments, single cells were marked as a region of interest using the MetaFluor software. Confocal images were analysed for intensity over the plasma membrane in the green  and red channel (Caveolin1) using ImageJ. For each protein of interest, five images were analysed. Values were normalized to the highest value and presented as per cent of maximal intensity in each channel. Intensity profiles were mapped using GraphPad Prism 6.07.The statistical significance of variance between two means was calculated using Origin Pro8.5 . Student's 2+]i  from 186 ± 4 nM upon application of ATP alone . The second phase was abolished in RB-2 exposed adipocytes.We investigated the effect of extracellularly applied ATP on 3T3-L1 adipocyte [Ca2+]i . Applica present . The fas present , whereasau level . In the cleotide . Pre-treTP alone  to 58 ± 2+. The initial [Ca2+]i transient was inhibited by 30%  and the stable elevation was abolished in the absence of extracellular Ca2+  inhibitor thapsigargin (1 µM during 30 min) to deplete intracellular calcium stores. The ATP response was absent from thapsigargin pre-treated cells . This decrease is likely due to ATP-dependent activation of plasma membrane Ca2+ pumps i peak. The store depletion subsequently activates influx of Ca2+ from the extracellular space, observed as a stable [Ca2+]i elevation.Our results suggest that ATP triggers release of Ca2+]i peak of a similar magnitude as that produced by ATP; Δpeak values were 199 ± 10 and 198 ± 9 nM, respectively.We investigated the gene expression of P2Y receptors (the P2ry gene family) in undifferentiated and differentiated 3T3-L1 adipocytes . In agre2+]i increases via the PLC/IP3 pathway. IP3 activates ER-localized IP3 receptors that work as Ca2+ channels thus leading to depletion of ER Ca2+ stores [2+]i averaged 47 ± 5 nM) and inhibited the sustained [Ca2+]i elevation. ATP remained capable of elevating [Ca2+]i in adipocytes pre-treated with U73343, a structurally close analogue to U73122 with no demonstrated effect on PLC. The Δpeak [Ca2+]i elevation produced in U73343-exposed adipocytes averaged 171 ± 15 nM . 2-APB, SKF96365 and Gd3+ all inhibited the [Ca2+]i increase observed upon reintroduction of Ca2+. These effects occurred in a concentration-dependent manner  induced a Δpeak [Ca2+]i of 95 ± 4 nM (n = 97), a significantly lower magnitude than that produced by ATP under the same conditions . This slower response is because thapsigargin inhibition of ER Ca2+ refilling results in gradual Ca2+ leakage, whereas ATP induces rapid Ca2+ depletion via activation of IP3 receptors [2+ was released under both conditions (337 ± 12 nM min with thapsigargin and 361 ± 17 nM min with ATP). The time to [Ca2+]i peak and the reversal to [Ca2+]i baseline were largely delayed upon addition of thapsigargin when compared with ATP ([Ca2+]i peak observed at 3.4 ± 0.3 min upon addition of thapsigargin and at 0.33 ± 0.02 min in response to ATP). Reintroduction of Ca2+ elevated [Ca2+]i of a magnitude comparable to that measured in the ATP experiments . In keeping with the ATP experiments, Gd3+ inhibited the elevation caused by the reintroduction of Ca2+ in a dose-dependent fashion, thus resembling that observed when Ca2+ stores were depleted by ATP  and its in SOCE . We thusStim1 or Orai1  or with a scramble control. Owing to the suggested role of TRPC1 in SOCE, we also tested the effect of Trpc1 knockdown. As shown in Trpc1 by ∼50% and that of Stim1 and Orai1 by ∼70% compared with the scramble control. The reduced expression of Stim1 resulted in a slight up-regulation of Trpc1 and Orai1 mRNA levels . Transfection with the alternative siRNA sequences reduced the expression of Stim1 by 55% and that of Orai1 by 65% compared with the scramble control (not shown). We again measured the increase in [Ca2+]i upon reintroduction of Ca2+ to thapsigargin-exposed cells. As shown in 2+]i elevation was, in agreement with data in Stim1 or Orai1 siRNA compared with scramble siRNA controls. Notably, the magnitude of maximum Ca2+ influx upon reintroduction of extracellular Ca2+ (∼80 nM) was in this experimental series in a range between that measured in 2+ storage/dynamics. In conclusion, our knockdown experiments confirm the presence of SOCE in white adipocytes and propose that STIM1 and ORAI1 are the chief components.The pharmacology of SOCC inhibitors is complex and they are known to affect targets other than those involved in SOCE . Thus, tA levels . We meassigargin . Surpris2+ permeable channel ORAI1 and we show the regulation of SOCE by external ATP. Ca2+ entry via store-operated channels has previously been shown in pre-adipocytes [2+ influx has also been demonstrated to negatively affect pre-adipocyte differentiation and adipogenesis [The findings described here demonstrate the functional existence of SOCE in white adipocytes. We present evidence of the presence of the two main molecular players involved in SOCE: the ER-localized calcium sensor STIM1 and the plasma membrane Caipocytes  and STIMogenesis . However2+]i via binding to P2Y2 receptors and activation of the PLC/IP3 pathway. WAT is abundantly innervated by the sympathetic nervous system and adrenergic nerve terminals co-release ATP with norepinephrine i in several cell types [2+]i has been shown to involve activation of both P2Y1 i interval has been shown to be necessary to support the insulin action. A basal [Ca2+]i is required for insulin-stimulated glucose uptake  at the sites of SOCCs specifically acts to regulate the secretion of hormone-containing vesicles that are stored in close vicinity to the plasma membrane [vs localize to exocytotic sites in other endocrine cell types [Evidently, Cay others  and by oipocytes –61, but ipocytes  suggestsnditions . Thus, lnditions , SOCE li+ stores , an intemembrane . This isll types ,63. Of pll types ,64 sincell types ,66.Drosophila adiposity [Stim1 are down-regulated in adipocytes isolated from obese and diabetic mice , thus suggesting that adipocyte SOCE may be disturbed also in mammalian metabolic disease. The recently emphasized importance of communication between adipose tissue and the brain [During the last two decades, it has become apparent that disturbed SOCE signalling is connected to a multitude of pathological conditions  includindiposity . Prelimihe brain  further