Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.Alteration of the nuclear Ca During a cardiomyocyte contraction, cardiac myocyte membrane depolarization causes the voltage-dependent L-type Ca2+ channel (LTCC) to drive Ca2+ entry into the cytoplasm2+ concentration triggers a greater release of Ca2+ from the sarcoplasmic reticulum (SR). The excess Ca2+ binds to the myofilament and induces the subsequent myocyte contraction. During cardiomyocyte relaxation, cytoplasmic Ca2+ is exported outside the cell via the sarcolemmal Na+/Ca2+ exchanger (NCX) and pumped into SR through the SR Ca2+ ATPase (SERCA). In addition to the role in contraction, intracellular Ca2+ has a definitive function as a secondary messenger for various cellular physiological processes. The increased Ca2+ level stimulates Ca2+-calmodulin (CaM)-dependent protein phosphatase 2B , which activates nuclear factor of activated T cells (NFAT) to drive transcription of several genes2+ signal also activates calmodulin-dependent kinases (CaMKs), which positively regulate several transcription factors including myocyte-enhancing factor 2 s (MEF2s) and mediate nuclear translocation of chromatin-modifying enzymes including histone deacetylases (HDACs)52+ in cardiomyocytes; these pathways are intimately involved in development, physiology, and pathophysiology of the heart. Nonetheless, it is unclear how oscillations in Ca2+ regulate signaling pathways, e.g., peak Ca2+, diastolic Ca2+, and micro location2+ oscillation in the nucleus (just as in the cytoplasm) is also observed in cardiomyocytes2+ transient is partly independent of the cytoplasmic Ca2+ transient in cardiomyocytes2+ transient102+ transient is an early event in cardiac remodeling2+ transient is finely regulated and plays an important role in cardiac pathophysiology, but it remains largely unclear how the nuclear Ca2+ transient is regulated in cardiomyocytes.Intracellular Ca2+ regulation, we focused on a specific molecule among the nuclear membrane proteins. We also focused on a genetic disease that is caused by a mutation in a nuclear membrane-associated gene and has a specific cardiac phenotype. Emerin (encoded by EMD) is ubiquitously expressed and located in the inner nuclear membrane141517182+ signaling is highly important for homeostasis within a contracting muscle, e.g., skeletal myocytes and cardiac myocytes. Disruption of Ca2+ signaling in cardiomyocytes results in cardiac hypertrophy and heart failure202+ transient. Therefore, we hypothesized that emerin is involved in nuclear Ca2+ signaling in cardiomyocytes and participates in the pathogenesis of some cardiac diseases.To understand the molecular mechanism of nuclear Ca2+ transient in neonatal rat ventricular cardiomyocytes (NRVCs) after hypertrophic stimuli and in EDMD-induced pluripotent stem (iPS) cell-derived cardiomyocytes. The hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in NRVCs. An Emd knockdown also increased the nuclear area, decreased the nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient without hypertrophic stimuli in NRVCs. Hypertrophic-stimulus-dependent changes in nuclear invagination and Ca2+ transients were attenuated by the Emd knockdown. The iPS cells were derived from a patient with EDMD and were induced to differentiate into cardiomyocytes. EDMD-iPS cell-derived cardiomyocytes also showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. Furthermore, an autopsied heart from a patient with EDMD showed an increase in nuclear area and a decrease in nuclear invagination. These data suggest that emerin has a crucial function in nuclear structure and in the nuclear Ca2+ transient in cardiomyocytes. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD.We analyzed nuclear structure and the nuclear Ca2+ indicator mag-fluo-4 can reveal the nuclear envelop and tubular invaginations that traverse the nucleus, which imply that the nuclear envelop and its tubular invaginations are functional Ca2+ stores. MitoTracker Red specifically accumulated in both the subsarcomeric mitochondria (located around the nucleus) and in the intermyofibrillar mitochondria, and the fluorescence intensity of cardiomyocytes is known to be much stronger than that of nonmyocytes26Nuclear size and shape are important for cellular fine structure and function. Altered nuclear size and shape are associated with a wide variety of pathologies222+ transient plays important roles in the pathogenesis of heart diseases, it is mostly unclear whether the nuclear Ca2+ transient in cardiomyocytes would be affected by various hypertrophic stimuli. To analyze the kinetics of nuclear and cytoplasmic Ca2+ transients, NRVCs were loaded with the Ca2+ indicator Fluo-4, which diffuses both to the cytoplasm and nucleus, and rapid resolution confocal microscopic analysis was then conducted282+ indicator, Rhod4 which had different Kd from fluo4, and observed similar nuclear Ca2+ dynamics . We performed immunostaining for LAMIN A/C to elucidate the nuclear morphology and immunostaining for cTnT to identify the cardiomyocytes . The nuc2+ has several physiological functions in cardiomyocytes. Cardiac contraction is finely regulated by cytoplasmic Ca2+: EC coupling. Depolarization of the plasma membrane in cardiomyocytes activates LTCC to open in order to increase Ca2+ influx into the cytoplasm, but intracellular Ca2+-dependent inactivation limits Ca2+ influx during an action potential2+ binds to the ryanodine receptor (RyR) in SR adjacent to the plasma membrane, and this event triggers a massive efflux of Ca2+ from the SR into the cytoplasm: Ca2+-induced Ca2+ release (CICR)2+ is sequentially removed from the cytoplasm. It seems that cytoplasmic Ca2+ is synchronously changed in the whole cytoplasm, but cytoplasmic Ca2+ is finely and locally regulated with respect to each microcompartment within the cell.Cytoplasmic Ca2+ transient shows different kinetics from those of the cytoplasmic Ca2+ transient2+ 362+ around the nucleus2+ signals independently of cytoplasmic Ca2+ 2+ from the cytoplasm surrounding the nucleus into the NE2+ homeostasis in the nucleoplasm282+ is also controlled by several nuclear membrane proteins, such as RyRs and nicotinic acid adenine dinucleotide phosphate receptors (NAADPRs)41The nuclear Ca2+ signaling2+ pump back to cytoplasm12452+. In cardiomyocytes, nuclear invagination is firstly changed during cardiac remodeling, which suggests that nuclear invagination may play a critical role in hypertrophic gene program activation and cardiac remodeling192+ pump back function mediated by nuclear invagination, rather than the dysregulation of Ca2+ release mechanism such as IP3 receptors and RyRs. In our experiments, the decrease in nuclear invagination may disturb the prompt Ca2+ pump back and prolong the half-decay time. The intracellular Ca2+ dynamics activates several transcription factors and co-factors, such as HDAC5, NFAT and MEF2C, which induce cardiac hypertrophy46482+ transient is an early event in cardiac remodeling2+ transient, alteration of the nuclear Ca2+ transient and HDAC5 activation were observed2+ which we observed would indicate the increased nuclear Ca2+ for a prolonged time in diastolic phase. The increased nuclear Ca2+ would activate Ca2+ dependent-transcription factors and co-factors in nucleus.The cellular environment affects the nuclear envelop shape including nuclear invagination2+ transient. EDMD is also caused by gene mutations in other nuclear-membrane-associated proteins, such as LMNA, SYNE1, SYNE2, SUN1, SUN2, and TMEM435253542+ transients and what happens next to the defect of the nuclear Ca2+ transient. These issues should be clarified in the future. We also observed the increase of nuclear size. Nuclear size is regulated by several mechanisms such as nucleocytoplasmic transport, nuclear structural components, and post-mitotic nuclear assemblyNumerous proteins have been identified in the NE membrane, including both ubiquitous and potentially cell type-specific proteins13Human cardiomyocytes from patients with genetic disorders are an ideal tool for clinical research, but their use is limited in basic research. Most basic researches conduct studies using animal models for research into relevant diseases. It should be noted that there are many differences between animals and humans, and some human diseases cannot be reproduced in animal models. Here, we used iPS cell-derived cardiomyocytes to elucidate the cellular pathophysiology of the patient with EDMD. Recent methodological and technological progress can help researchers to derive iPS cells from patients with genetic disorders, induce iPS cells to differentiate into cardiomyocytes, and then to purify the cardiomyocytes5859606162633031322+ transient is affected by hypertrophic stimuli in NRVCs. The nuclei of Emd knockdown NRVCs also show characteristics similar to those of hypertrophic-factor-stimulated cardiomyocytes, but cell size and the cytoplasmic Ca2+ transient are not affected by the Emd knockdown. Human EDMD-iPS cell-derived cardiomyocytes also show attenuated nuclear invagination and an altered nuclear Ca2+ transient. These data suggest that EDMD would be caused by disturbances of nuclear invagination and of the nuclear Ca2+ transient. Nuclear invagination and the nuclear Ca2+ transient may be therapeutic targets in EDMD and cardiac remodeling. In addition, emerin may be a molecular mediator of nuclear invagination and of the nuclear Ca2+ transient in the heart.In summary, nuclear invagination is attenuated and the nuclear CaPrimary cultures of NRVCs were prepared as described previously4 0.33 mM, MgCl2∙6H20 0.53 mM, CaCl2∙2H20 1.8 mM, HEPES-NaOH 5 mM, Glucose 5.5 mM) for 30 min at 37 °C. A further 30 min was allowed for deesterification. Ca2+ imaging was performed at room temperature by confocal microscopy. Ca2+ imaging was performed by confocal microscopy  using a ×40/0.75 NA. The pinhole was set to 1 Airy unit, resulting in optical-slice thickness of 1.0 μm. The confocal plane was set to the middle (z-axis) of the nuclei. A 512-pixel scan line was positioned to include the nucleus, and scanning was performed every 2 ms . Cardiomyocytes spontaneously beating and electrically stimulated at 0.25 Hz were analyzed for F/F0, time to peak, and half-decay time.Cardiomyocytes were placed in 35-mm glass-bottom dishes and loaded with 5 μM Fluo-4 (Invitrogen) or Rhod4 (AAT bioquest) in Tyrode’s solution , endothelin 1 (Sigma-Aldrich), and phenylephrine (Sigma-Aldrich) were used for the drug assays.2+ imaging.Rat Emd siRNA (s129800) and scrambled siRNA (4390846) were purchased from Life Technologies. SiRNA was diluted with OPTI-MEM (Invitrogen) to 20 nmol/L and added in the amount of 125 μL to 7.5 μL OPTI-MEM-containing LipofectamineRNAi Max (Invitrogen). After incubation for 10 min at room temperature, the siRNA-Lipofectamine mixture was added to NRVCs with subsequent incubated for 24 hours at 37 °C without horse serum and antibiotic-antimycotic agents. The cells were transferred to a maintenance medium without the horse serum and antibiotic-antimycotic agents, and then 100 nmol/L ET-1, 100 nmol/L Ang II, or 50 μmol/L phenylephrine were added with subsequent incubation for 48 hours. The cells were then harvested for RNA analysis, immunofluorescence staining, and CapCAG-GFP-IRES-Emerin plasmid was constructed by emerin cDNA and pCAG-GFP (Addgene) plasmid. Each 2.5 μg plasmid were diluted with 150 μl OPTI-MEM (Invitrogen) and added in the amount of 125 μL to 3.5 μL OPTI-MEM-containing Lipofectamine3000 (Invitrogen). After incubation for 10 min at room temperature, the mixture was added to NRVCs with subsequent incubated for 48 hours at 37 °C without horse serum and antibiotic-antimycotic agents. The cells were transferred to a maintenance medium without the horse serum and antibiotic-antimycotic agents, and then 50 μmol/L phenylephrine were added with subsequent incubation for 48 hours. The cells were then harvested for immunofluorescence staining.® qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative RT-PCR for emerin (EMD) and GAPDH genes was conducted by means of the Power SYBR® Green PCR Master Mix (Applied Biosystems). Quantitative RT-PCR for EMD and GAPDH was performed with the primer set shown in the For quantitative RT-PCR assays, total RNA was extracted from NRVCs (after 4 days of culturing) by means of the TRIzol Reagent (Invitrogen). One microgram of total RNA was reverse-transcribed into cDNA with the ReverTra AceProteins from self-beating embryoid bodies (EBs) were extracted with RIPA buffer . The proteins were loaded on Mini-PROTEAN TGX Gels  and then electrophoretically separated and transferred onto nitrocellulose blotting membranes . The primary antibodies were anti-emerin (Abcam), anti-SYNE1 (Millipore), and anti-GAPDH . Protein expression was visualized with appropriate horseradish peroxidase-conjugated secondary antibodies  and enhanced chemiluminescence , and was detected using an LAS-3000 luminoimager . Protein bands were quantified using the ImageJ software .Colonies of undifferentiated iPS cells plated on a layer of mouse embryonic fibroblasts (feeder cells), iPS cell-derived cardiomyocytes, or NRVCs plated on fibronectin-coated dishes were fixed with 4% paraformaldehyde  for 30 min at 4 °C. After that, the cells were permeabilized with 1% Triton X-100 and blocked with ImmunoBlock . The samples were incubated at 4 °C overnight with each of the following primary antibodies: anti-OCT3/4 , anti-NANOG , anti-SSEA3 , anti-SSEA4 (Millipore), anti-Tra1-81 (Millipore), anti-Tra1-60 (Millipore), anti-troponin T , anti-α-actinin (Sigma-Aldrich), anti-NKX2.5 (Santa Cruz Biotechnology), anti-HCN4 (Abcam), anti-emerin , anti-LAMIN A/C (Santa Cruz Biotechnology), anti-SUN1 (Abcam), anti-LBR(Abcam), anti-NPC(Abcam) and anti-SYNE1 (ATLAS). Preparations were incubated for 1 hour at room temperature with the isotype-specific secondary antibodies: an Alexa Fluor 488-conjugated chicken anti-rabbit IgG antibody, Alexa Fluor 594-conjugated goat anti-mouse IgG antibody, Alexa Fluor 488-conjugated goat anti-rat IgM antibody, Alexa Fluor 594-conjugated goat anti-mouse IgM antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG antibody, Alexa Fluor 594-conjugated chicken anti-goat IgG antibody, Alexa Fluor 633-conjugated goat anti-mouse IgG antibody and an Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody, which were all acquired from Invitrogen. Nuclei were counterstained with 50 ng/mL 4′,6-diamidino-2-phenylindole  or 1 μg/mL Hoechst 33258 (Lonza). Fluorescent signals were detected under a fluorescence laser microscope  with ×10/0.45 numerical aparature (NA)or a confocal microscope with ×40/0.75 NA, ×63/1.2 NA or a ×63 × 3/1.2 NA oil-immersion objective .The cytoplasm area was assessed by means of troponin T-positive area obtained by confocal microscopy (LSM5 DUO at ×40 × /0.75 NA), and the nuclear area was assessed by means of the DAPI-positive area set to the middle of a visual field under the LSM5 DUO at ×63 × 3/1.2 NA oil-immersion objective. Analysis of individual cells was performed in a blinded experiment using the area calculating system of LSM510. We performed three dimensional images with 0.35 μm z-stack thickness to assess nuclear invagination. Nuclear volume was assessed with DAPI staining by the IMARIS software . Nuclear invagination was assessed with LAMIN A/C or LBR as previously describedAll subjects provided informed consent for blood testing for genetic aberrations associated with EDMD. The Ethics Committee of Kanazawa University approved the isolation and use of patients’ and control somatic cells for iPS cell experiments , which were performed only after the patients and controls had provided written informed consent. Isolation and use of patients’ and control cells were approved by the Ethics Committee of Keio University (20-92-5) and were performed after written informed consent was obtained. The study was performed conforming to the Declaration of Helsinki.6/well to a fresh anti-CD3 antibody-coated 6-well plate, and incubated for an additional 24 hours. Then, solutions containing the Sendai virus vectors individually carrying each of OCT3/4, SOX2, KLF4, and MYC were added at multiplicity of infection (MOI) of 10. After 24 hours of infection, the medium was changed to a fresh KBM 502 medium , and the cells were collected and split at 5 × 104 cells into 10-cm plates preseeded with mouse embryonic fibroblasts. After additional 24 hours of incubation, the medium was changed to the hiPSC medium (described below) supplemented with 4 ng/mL basic fibroblast growth factor (bFGF). Approximately 20 to 30 days after the infection, iPS cell colonies appeared and were picked. Sendai virus vectors carrying the same four specific transcriptional factors (separately) were purchased from Life Technologies (CytoTune-iPS Reprogramming Kit).To study the role of emerin in human cardiomyocytes, we focused on the human disease that is associated with a mutation in EMD58All subjects provided informed consent for blood testing for genetic aberrations associated with EDMD. DNA sequencing was used to confirm the presence of the relevant mutation in patient-derived iPS cells. Genomic DNA was isolated using the Gentra Puregene Cell Kit , and the region encoding the mutation was amplified by PCR. The PCR product was analyzed by electrophoresis in a 1% agarose gel and purified using a Wizard SV Gel and PCR Clean-Up System . The purified PCR product was sequenced with the original primers shown in the Human iPS cells were maintained for several passages on irradiated mouse embryonic fibroblast feeder cells in the hiPSC culture medium: 80% of DMEM/F12 , 20% of the Knockout Serum Replacement , 4 ng/mL bFGF , 2 mmol/L L-glutamine (Invitrogen), 0.1 mmol/L nonessential amino acids (Sigma-Aldrich), 0.1 mmol/L 2-mercaptoethanol, 50 U/mL penicillin, and 50 mg/mL streptomycin (Invitrogen). The hiPSC medium was changed every 2 days, and the cells were passaged using 1 mg/mL collagenase IV (Invitrogen) every 5–7 days. After several passages, these cell lines were cultured in the human iPS cell medium consisting of Repro Stem (Repro cell) and 50 U penicillin and 50 mg/mL streptomycin (Invitrogen).2+ imaging analyses, a single-cell suspension of cardiomyocytes (isolated from spontaneously contracting EBs) by means of 0.25% trypsin (Invitrogen), 1 mg/mL type IV collagenase (Invitrogen), and ADS buffer  for 30 min. at 37 °C. The cells were plated in fibronectin-coated dishes and incubated for 5 days in the presence of 20% FBS. To analyze the iPSC-derived cardiomyocytes, the mixtures of contracting EBs derived from two independent iPSC lines were used in each case.The iPSCs were harvested using 1 mg/mL collagenase IV, and transferred to ultra-low attachment plates  in the Repro stem medium supplemented with 10 ng/mL BMP4 (Wako) and 5 μmol/L blebbistatin (Sigma) from day 0 to day 1. The medium was changed to Stempro34 (Gibco) supplemented with 50 μg/mL ascorbic acid (Sigma), 2 mmol/L glutaMAX (Invitrogen), 10 ng/mL BMP4, and 25 ng/mL activin A from day 1 to day 3. On day 3, the medium was changed to Stempro34 supplemented with 50 μg/mL ascorbic acid and 2 mmol/L glutaMAX. On day 4.5, the medium was changed to Stempro34 supplemented with 50 μg/mL ascorbic acid, 2 mmol/L glutaMAX, and 2.5 μmol/L IWR-1 (Sigma). On day 8.5, the medium was changed to the differentiation medium consisting of α Minimum Essential Medium (Gibco) supplemented with 2 mmol/L L-glutamine (Invitrogen), 0.1 mmol/L nonessential amino acids (Sigma), 0.1 mmol/L 2-mercaptoethanol, 50 U/mL penicillin, and 50 mg/mL streptomycin (Invitrogen), and 10% of fetal bovine serum (Gibco). The medium was replaced every second day or every third day. After 20 to 30 days of differentiation, self-contracting EBs were picked and placed into fresh ultra-low attachment plates (Corning). For immunohistochemical and CaHistological sections of the autopsied heart from an 83-year-old patient with EDMD and a 78-year-old control patient were permeabilized with 1% Triton X-100 after deparaffinization, and the antigens were activated with Histo VT one  at 90 °C for 40 min. After that, cardiomyocyte-containing tissue slices were blocked with ImmunoBlock  at 4 °C overnight and were incubated at 4 °C overnight with each of the following primary antibodies: anti-LAMIN A/C (Santa Cruz Biotechnology) and anti-troponin T . Preparations were incubated for 1 hour at room temperature with the isotype-specific secondary antibodies: an Alexa Fluor 488-conjugated chicken anti-rabbit IgG antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG antibody, both obtained from Invitrogen. Nuclei were counterstained with 50 ng/mL DAPI (Invitrogen). Fluorescent signals were detected using a fluorescence laser microscope  or by confocal microscopy with a ×40/1.2 NA or ×63 × 3/1.2 NA oil-immersion objective  just as during the measurement of the cardiomyocyte cytoplasmic and nuclear area.Continuous data are presented as mean ± SD. The significance of differences between two means was evaluated by unpaired t tests. Fisher’s exact probability test was used for comparisons between groups. Comparisons between more than two groups involved analysis of variance (ANOVA) followed by Dunnett’s test. Categorical data are presented as mean ± SE and are compared by using Chi-squared test. All analyses were conducted in EZRHow to cite this article: Shimojima, M. et al. Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient. Sci. Rep.7, 44312; doi: 10.1038/srep44312 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through “hyperactive signaling junctions,” wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals.In human aortic smooth muscle cells, prostaglandin E The versatility of these messengers depends on both the spatiotemporal organization of the changes in their concentration within cells (2+ concentration ([Ca2+]i) stimulate contraction, but receptors that stimulate formation of cAMP usually cause relaxation. The clinical importance is clear from the widespread use of β-agonists to provide symptomatic relief from asthma  are two important inflammatory mediators. Their effects on VSM, which include regulation of proliferation . The initial response is mediated by Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3R) and it is followed by Ca2+ entry across the plasma membrane  and substantially attenuates the Ca2+ signals evoked by histamine. Here, we show that inhibition of histamine-evoked Ca2+ signals by PGE2 is mediated by cAMP delivered within “hyperactive signaling junctions.” The response does not require EPACs, but it is attenuated by inhibition of PKA. The effect of PGE2 on histamine-evoked Ca2+ signals does not result from a decrease in IP3R sensitivity or from increased Ca2+ extrusion from the cytosol, nor does PGE2 affect the Ca2+ signals evoked by stimulation of either endogenous type 1 protease-activated receptor (PAR1) or heterologously expressed muscarinic M3 acetylcholine receptors. We suggest that PKA uncouples H1 histamine receptors from the guanine nucleotide-binding protein, Gq/11, and activation of phospholipase C (PLC). Our results establish that digital signaling from PGE2 receptors, through cAMP and PKA, inhibits histamine-evoked Ca2+ signals.We demonstrated previously that histamine, through Hmembrane . PGE2, aSp-cAMPS, 6-Bnz-cAMP, 8-pCPT-2′-O-Me-cAMP, Rp-cAMPS, Rp-8-CPT-cAMPS, ESI-09, and HJC0197 were from Biolog . Ionomycin, SQ 22536, DDA and myristoylated-PKA inhibitor peptide (PKI) were from Merck-Millipore . A membrane-permeant peptide inhibitor of A kinase-anchoring proteins (AKAPs) [stearated Ht31 AKAP inhibitor peptide (st-Ht31)] and its proline-modified inactive form (st-Ht31P) were from Promega . Thapsigargin was from Alomone Laboratories . PAR1 peptide, histamine dihydrochloride, forskolin, IBMX, and PGE2 were from Sigma-Aldrich . Butaprost (free acid) and L902,688 were from Cayman Chemicals . Membrane-permeant caged IP3 (ci-IP3PM) was from SiChem .  adenine ci-IP3PM, d-2,3-O-isopropylidene-6-O-benzyl-myo-inositol 1,4,5-trisphosphate-hexakis(propionoxymethyl) ester was from Perkin Elmer . Fluo-8 was from Stratech Scientific Ltd . Other reagents were from Sigma-Aldrich, sources specified previously . eviously  or identHuman ASMC from the American Tissue Culture Collection  or Dr. Trevor Littlewood  were cul2+]i were recorded from cell populations using confluent cultures of ASMC grown in 96-well plates and loaded with Fluo-4 or Fluo-8. Experiments were performed in HEPES-buffered saline (HBS) at 20°C. HBS had the following composition (mM): NaCl 135, KCl 5.9, MgCl2 1.2, CaCl2 1.5, glucose 11.5, and HEPES 11.6 (pH 7.3). Fluorescence was recorded using a FlexStation 3 fluorescence plate-reader  and calibrated to [Ca2+]i as described  were loaded with Fura-2 in HBS containing Fura-2 AM (4 μM), probenecid (2.5 mM), and pluronic F127 (0.02% v/v) for 1 hour at 20°C. Fluorescence  was recorded using an Olympus IX71 inverted fluorescence microscope and Luca (electron-multiplying charge-coupled device) EMCCD Andor Technology, Belfast, UK camera. After correction for background fluorescence, determined by addition of ionomycin (1–5 μM) in HBS containing MnCl2 (1 mM), fluorescence ratios (F340/F380) were calibrated to [Ca2+]i . After 30 minutes on ice, 3H-cAMP was separated from other 3H-labeled adenine nucleotides (Confluent cultures of ASMC grown in 24-well plates and labeled with leotides .Plasmids encoding PKI (pRSV-PKI-v2) and its inactive form (pRSV-mut PKI-v2) were from Addgene  ; they wel-lysine-coated imaging dishes  were loaded  with a membrane-permeant form of caged IP3  in HBS with probenecid (2.5 mM) and pluronic F127 (0.02% v/v). Fluo-4 AM (4 μM) was then added and after 45 minutes at 20°C, the medium was replaced with HBS containing only probenecid. After a further 45 minutes, this medium was replaced with HBS. Cells were illuminated with a 488-nm diode-based solid-state laser, and emitted fluorescence (500–550 nm) was captured with an EMCCD camera. Three UV flashes  from a JML-C2 Xe flash-lamp  allowed photolysis of caged IP3 (ci-IP3). Responses are reported as F/F0, where F0 and F are fluorescence intensities corrected for background recorded from the same region of interest immediately before (F0) and after stimulation (F).Confluent cultures of ASMC grown on poly-d-myo-[2-3H]-inositol (10 μCi/ml) for 48 hours at 37°C. After washing, cells were incubated at 20°C in HBS with LiCl (10 mM) for 5 minutes before stimulation. Reactions were terminated by aspirating medium and adding cold HClO4  containing phytic acid (0.2 mg/ml). After 30 minutes, the acid-extract was removed, the cells were scraped into 50 mM Tris at pH 7 (400 μl), and the pooled extract and cells were centrifuged . The supernatant was neutralized using K2CO3 (1 M) with EDTA (5 mM). 3H-inositol phosphates were separated using ion-exchange columns.ASMC in 12-well plates were cultured until confluent. The medium was then supplemented with 3 mass, ASMC in 75-cm2 flasks were stimulated, the medium was removed, and the incubations were terminated by scraping cells into ice-cold ethanol (1 ml). After 30 minutes, extracts were dried and suspended in 300 μl of Tris-EDTA medium . Equilibrium-competition binding using 3H-IP3 , rat cerebellar membranes (10 μg) and cell extract (20–100 μl) in a final volume of 200 μl of TEM was used to determine the IP3 content of the extracts , protease inhibitors , and phosphatase inhibitors . Scraped cells were disrupted by ∼30 passages through a 28-gauge needle and sonicated (3 × 10 seconds). Proteins were separated by SDS-PAGE  and transferred to a polyvinylidene difluoride membrane . Membranes were washed (5 minutes) with Tris-buffered saline , blocked by incubation in TBS containing 0.1% Tween-20 (TBS-T) and 5% (w/v) nonfat milk powder , and then washed with TBS-T (3 × 5 minutes). Blots were incubated for 12 hours at 4°C with primary antibody (1:1000) in TBS-T with 5% (w/v) BSA. After washing (3 × 5 minutes), blots were incubated with horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody  for 1 hour in TBS-T with 5% (w/v) nonfat milk powder. After further washing (3 × 5 minutes), bands were detected using ECL Prime  and quantified using GeneTools . The primary rabbit antibodies recognize PKA-phosphorylated sequences RXXS*/T*  and vasodilator-stimulated phosphoprotein  phosphorylated at Ser-157 (New England Biolabs) or VASP clone 43 .Confluent ASMC in 75-cmQPCR was performed as described  using pr2 varied between patients and with cell passage. Results are, therefore, often presented as normalized values  or as differences between paired comparisons . Two-tailed paired or unpaired Student’s t test, or one-way analysis of variance followed by Bonferroni’s test, were used as appropriate.Concentration-effect relationships were individually fitted by nonlinear curve-fitting to Hill equations . The absolute sensitivities and amplitudes of the responses to histamine and PGE2 inhibits the Ca2+ signals evoked by histamine in human ASMC. Most experiments were performed at 20°C to minimize loss of the cytosolic Ca2+ indicator. However, in parallel analyses we confirmed that a maximally effective concentration of PGE2 (10 μM) caused indistinguishable inhibition of the Ca2+ signals evoked by histamine (100 μM) whether the analyses were performed at 20°C  or 37°C (45% ± 6%). In parallel measurements of the effects of PGE2 on intracellular cAMP and histamine-evoked Ca2+ signals, the cAMP response   . Pretreaistamine ; Table 1istamine . Maximaladditive . These r2+ signals by reducing the maximal response and the sensitivity to histamine .High concentrations of 8-Br-cAMP also inhibited histamine-evoked Caistamine . 8-Br-cAr stores . The effμM, 20 minutes) did not prevent the inhibition of histamine-evoked Ca2+ signals by PGE2 (μM) of either antagonist abolished the Ca2+ signals evoked by histamine (data not shown). We have not explored this effect further, although the antagonists caused similar inhibition of carbachol-evoked Ca2+ signals in human embryonic kidney 293 cells  partially inhibited Ca2+ signals evoked by a submaximal concentration of histamine, but the maximal inhibition was less than half that evoked by PGE2 or 8-Br-cAMP  was less than that caused by PGE2 (56% ± 3%)  . More im of IBMX , demonst3-evoked Ca2+ release, nor are EPACs responsible. We therefore assessed the role of PKA.Most effects of cAMP are mediated by PKA, EPACs, or cyclic nucleotide-regulated plasma membrane cation channels . The lat2 or 8-Br-cAMP stimulated similar levels of protein phosphorylation in ASMC and their effects were nonadditive (′-O-Me-cAMP (2-evoked protein phosphorylation was attenuated by inhibition of either PKA (with H89) or AC [with 1 mM SQ 22536 with 200 μM DDA (SQ/DDA)] ] .2 and 8-Br-cAMP caused phosphorylation of the same proteins .Maximal concentrations of PGEproteins , but the by PGE2 . Convers-Br-cAMP . Similar2 may be exposed to high local concentrations of cAMP, which might then effectively compete with the inhibitor Rp-8-CPT-cAMPS. Conversely, PKA activated by 8-Br-cAMP, which would probably be uniformly distributed within the cell, may be more accessible to ATP than PKA activated by PGE2, and so less susceptible to inhibition by H89.These results suggest that PKA activated by PGE2+ signals by PGE2 or 8-Br-cAMP was inhibited by Rp-8-CPT-cAMPS (1 mM), which reduced the sensitivity to PGE2  and 8-Br-cAMP (ΔpIC50 = 0.6 ± 0.1) without affecting the maximal inhibition  also attenuated the inhibition of histamine-evoked Ca2+ signals by 8-Br-cAMP (ΔpIC50 = 1.13 ± 0.18 n = 3) and PGE2 . But using a baculovirus, we achieved expression of PKI in >90% of cells, and this caused 49% ± 6% (n = 3) inhibition of the VASP phosphorylation evoked by PGE2 (100 nM) . Expression of an inactive PKI (mut PKI) had no effect on PGE2-evoked protein phosphorylation . The effects of H89 and PKI on the inhibition of histamine-evoked Ca2+ signals by PGE2 were similar: Each substantially reduced the maximal inhibition without significantly affecting the IC50 for PGE2  and EP4  receptors were similar to those observed with PGE2 . We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 is mediated by cAMP and requires PKA  . This suggests that the attenuated Ca2+ signals do not result from PGE2 stimulating Ca2+ extrusion from the cytosol.The rate at which [Ca3 to activate IP3R directly in Fluo-4-loaded ASMC. Single-cell analyses of ASMC established that most cells  responded to histamine (1 mM) with an increase in [Ca2+]i, and that two successive challenges with histamine evoked indistinguishable Ca2+ signals , without significantly affecting the number of cells that responded   . These r3 responded to UV flashes with rapid increases in Fluo-4 fluorescence . The amplitudes of these signals were less than those evoked by a maximal concentration of histamine  was compared with the first response for each cell (R2/R1)  , or cell2/R1)    that is +), histamine  stimulated a small accumulation of 3H-inositol phosphates in ASMC. Although the response was modestly attenuated by PGE2 (10 μM), the effect was not statistically significant IP3, histamine stimulated IP3 accumulation, and the response was attenuated by PGE2, although the latter again failed to achieve statistical significance (3 formation in single cells using a fluorescence resonance energy transfer (FRET)-based IP3 sensor . Only thrombin reproducibly evoked substantial increases in [Ca2+]i. Thrombin is a protease that cleaves the type 1 protease-activated receptor (PAR1) to unmask an N-terminal ligand. Thrombin and the PAR1 peptide itself evoked concentration-dependent increases in [Ca2+]i in ASMC  attenuated the Ca2+ signals evoked by histamine without affecting those evoked by PAR1 peptide  increase in [Ca2+]i, with a maximal increase  comparable to that evoked by histamine  that might be expected to evoked Ca in ASMC . In para peptide . Althoug by PGE2 . After h13 nM, 2 . These r2-evoked cAMP formation   attenuated PGE, n = 4)  and prot, n = 4) . Howeverutaprost . Althoug signals . Howeveractivity ?2. Available antibodies do not allow quantitative assessment of the expression of AC subtypes, but QPCR analysis shows that human ASMC express similar amounts (∼30%) of AC3, AC7, AC9, some AC6 (∼10%), and detectable AC4 (∼2%) . AC9 probably does not mediate the effects of PGE2 on Ca2+ signals because AC9 is insensitive to forskolin and NKH 477 , the sensitivity to L902,688, a selective agonist of EP4 receptors, was modestly reduced by SQ/DDA   . This ob2+ signals. A membrane-permeant peptide that disrupts association of AKAPs with PKA (st-Ht31) but not its inactive analog (st-Ht31P), significantly attenuated the protein phosphorylation evoked by PGE2, but neither peptide affected the concentration-dependent inhibition of histamine-evoked Ca2+ signals by PGE2 . These results suggest that AKAPs are probably not important components of the signaling pathway from PGE2 to inhibition of Ca2+ signals.We considered whether AKAPs, which are widely implicated in assembling PKA with its regulators and effectors , might c3-mediated Ca2+ signals evoked by activation of H1 histamine receptors are attenuated by PGE2. Several lines of evidence show that this inhibition is mediated by cAMP. The concentration-effect relationships for regulation of AC and Ca2+ signals by PGE2 are consistent with cAMP lying upstream of Ca2+ in the signaling pathway  mediated by PKA . Nor would this mechanism be consistent with the lack of effect of PGE2 on the Ca2+ signals evoked by stimulation of endogenous PAR1 or heterologously expressed M3 muscarinic receptors (3-evoked Ca2+ release (3R1 and IP3R2) and cAMP (IP3R1-3) more often potentiate responses to IP3 . Our results do, however, reveal an additional complexity in the pathways linking PGE2 to inhibition of histamine-evoked Ca2+ signals. Although cAMP mediates this inhibition, the concentration-dependent effects of PGE2 were insensitive to substantial inhibition of AC (2 and EP4 receptors lead to the scheme shown in 3 formation is mediated by delivery of cAMP within signaling junctions. Activation of a junction allows local delivery of a supersaturating concentration of cAMP to PKA, allowing each junction to function as a robust on-off switch. We suggest that the concentration-dependent effects of PGE2 arise from recruitment of these junctions and not from graded activity within individual junctions. Such digital signaling from receptors to intracellular targets via hyperactive junctions (Cyclic AMP can be locally delivered to intracellular targets . AKAPs pon of AC . These runctions  allows runctions .


Salvia miltiorrhiza. This study aimed to investigate the effect of STS on ion transport in mouse tracheal epithelium and the mechanisms underlying it. Short-circuit current (Isc) was measured to evaluate the effect of STS on transepithelial ion transport. Intracellular Ca2+ imaging was performed to observe intracellular Ca2+ concentration ([Ca2+]i) changes induced by STS in primary cultured mouse tracheal epithelial cells. Results showed that the apical application of STS at mouse trachea elicited an increase of Isc, which was abrogated by atropine, an antagonist of muscarinic acetylcholine receptor (mAChR). By removing ambient Cl− or applying blockers of Ca2+-activated Cl− channel (CaCC), the response of STS-induced Isc was suppressed. Moreover, STS elevated the [Ca2+]i in mouse tracheal epithelial cells. As a result, STS stimulated Cl− secretion in mouse tracheal epithelium via CaCC in an mAChR-dependent way. Due to the critical role of Cl− secretion in airway hydration, our findings suggested that STS may be used to ameliorate the airway dehydration symptom in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD).Sodium tanshinone IIA sulfonate (STS) is a derivate of tanshinone IIA, a lipophilic compound in  A series of inhibitors including U73122 , 2-APB (50 μM) and thapsigargin  were used respectively and the peak values of the responses induced by STS , a cation-permeable Ca2+ channel, mediated the Ca2+ influx in the STS-induced elevation in [Ca2+]i. But the results showed that the STS-induced response was not decreased by application of the TRPV1 blocker capsazepine  i i . We obseSalvia miltiorrhiza on Cl− channel. The extract from the root of Salvia miltiorrhiza is reported to induce salivary fluid secretion by activating Na+-K+-Cl− cotransporter (NKCC) to maintain Cl− secretion, however, the involvement of Cl− channel has not been investigated in the research i  cytokine interleukin-13 [in vivo experiments are needed to investigate whether STS could ameliorate the airway dehydration symptom in cystic fibrosis and COPD. The effect of STS on airway epithelium in Th2 cytokines environment also needs to be considered carefully.Activation of CaCC by ATP or UTP released from airway secretory cells contributes to the optimized depth of the periciliary layer . The hydeukin-13 , 43. Thu− secretion via CaCC in an mAChR-dependent way in airway epithelium, which suggested a new application field of this traditional Chinese medicine monomer in respiratory system.Chinese herbal monomers possess many advantages, such as good quality, controllability and clear toxicology. Conclusively, this study demonstrated that STS induced ClS1 FigIsc. ΔIsc induced by STS  with the application of quinidine is 69.5 ± 13.7 μA/cm2 (n = 4). ΔIsc caused by STS  without quinidine is 71.9 ± 6.2 μA/cm2 (n = 9). Values are mean ± S.E.M. .Comparison of the effects of application of 1 mM quinidine at both apical and basolateral side of mouse trachea on STS  induced Δ(TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Strongylocentrotus purpuratus sperm flagella, triggers a signaling pathway that culminates with a train of intracellular calcium oscillations, correlated with changes in sperm swimming pattern. This pathway has been widely studied but not fully characterized. Recent work on Arbacia punctulata sea urchin spermatozoa has documented the presence of the Ca2+ CatSper channel in their flagella and its involvement in chemotaxis. However, if other calcium channels participate in chemotaxis remains unclear. Here, based on an experimentally-backed logical network model, we conclude that CatSper is fundamental in the S. purpuratus speract-activated sea urchin sperm signaling cascade, although other Ca2+ channels could still be relevant. We also present for the first time experimental corroboration of its active presence in S. purpuratus sperm flagella. We argue, prompted by in silico knock-out calculations, that CatSper is the main generator of calcium oscillations in the signaling pathway and that other calcium channels, if present, have a complementary role. The approach adopted here allows us to unveil processes, which are hard to detect exclusively by experimental procedures.Spermatozoa sea urchin swimming behaviour is regulated by small peptides from the egg outer envelope. Speract, such a peptide, after binding to its receptor in  S. purpuratus and Lytechinus pictus sea urchin spermatozoa respond to speract, a decapeptide present in the eggs’ external layer that diffuses into the sea. When speract binds to its receptor, which is located in sperm flagellar membrane, a signaling cascade initiates. This biochemical pathway is finely regulated by changes in membrane permeability to certain ions. The ensuing speract-activated signaling pathway (SASP) produces a train of intracellular Ca2+ concentration ([Ca2+]i) oscillations. These [Ca2+]i fluctuations are correlated with changes in the sperm swimming behaviour. Under two dimensional experimental conditions, sea urchin spermatozoa swim describing circles close to surfaces. When flagella detect increases in Ca2+ influx rate, their beating becomes asymmetric and the spermatozoon undergoes pronounced turns that are then followed by straighter swimming2.In their search for the egg, K+ channel (KCNG) that hyperpolarizes the membrane potential. Hyperpolarization influences several processes: i) it stimulates a K+/Na+/Ca2+ exchanger (NCKX)7 that decreases [Ca2+]i, ii) activates a Na+/H+ exchanger (NHE)6 which increases intracellular pH (pHi), iii) diminishes inactivation of high and low voltage-dependent calcium channels  and iv) stimulates a voltage-activated cAMP-dependent Na+ channel (HCN) that depolarizes6. The pHi elevation stimulates a soluble adenylyl cyclase (sAC)7 which synthesizes cAMP that in turn opens the previously mentioned HCN channel, as well as a cAMP-dependent Ca2+ channel (cAMPCC) that repolarizes the membrane. Via this chain of events, the cAMPCC acquires a pH dependence. Cyclic nucleotides levels are strongly controlled by phosphodiesterases (PDE) which degrade them. The cAMP dependence of this channel may be indirect. Repolarization allows the opening of LVA and HVA Ca2+ channels; these channels have been thought to be responsible for the [Ca2+]i fluctuations15. [Ca2+]i increases may open calcium-dependent K+ channels (CaKC) and chloride channels (CaCC). These last two channels can restart the pathway through a new hyperpolarization17. Calcium extrusion is carried out by the calcium pumps (CaP), and the above mentioned NCKX. The cyclic alternation of hyperpolarization and repolarization generates the train of oscillations in [Ca2+]i that govern the sea urchin sperm swimming pattern16. Despite that this SASP has been widely studied, many questions remain regarding participating elements and their relations, mainly due to experimental limitations.After speract binding, the speract-receptor complex activates a membrane guanylate cyclase (GC) which in turn produces cGMP, this second messenger opens a cGMP-dependent 23. The state of the network consists of a set of N discrete variables σ1, σ2, …, σn. Most of the variables take on two values, namely, 0 for closed/off and 1 for on/open states. However, an accurate description of the dynamical processes in the network required four nodes to be represented by three-state variables: the membrane potential ; the LVA and HVA Ca2+ channels ; and the intracellular calcium concentration ([Ca2+]i) . The state of each node σn is determined by its set of regulators, i.e. the nodes functionally linked to it. Let us denote as k regulators of σn. Then, at each time step the value of σn is given byFn is the regulatory function specific to that node. For the construction of these regulatory functions 25 may plays a role in the first stages of the pathway. Though the fine regulation of pHi via sNHE is not yet fully understood, the establishment of a direct link with [Ca2+]i fluctuations is appealing. A strong candidate for this link is the calcium channel CatSper, since in mammals it has been shown to be sperm specific28, activated by alkalization, mildly voltage-sensitive and essential for fertilization29. Additionally, the sequences that codify this channel have been found in S. purpuratus genome30. The participation of a pHi dependent Ca2+ channel in S. purpuratus sperm physiology was conjectured in ref. A. punctulata sperm flagella32. What remains to be shown is whether or not it is the principal generator of oscillations in the signaling pathway of S. purpuratus and if other calcium channels are accessory. If this were the case, CatSper would be the main controller of the [Ca2+]i oscillations and consequently of the sperm swimming behaviour.Experimental observations have shown that in 16. In that study, it was shown that an in silico blockage of the CaKC increased the time between subsequent calcium peaks as well as the average [Ca2+]i. However, when experiments with Iberiotoxin, a specific blocker of the CaKC channel33, were carried out, it was observed that though the time between calcium peaks of the speract-dependent [Ca2+]i oscillations increased, the average [Ca2+]i diminished. As a way out of this disagreement, the existence of other ionic channels in the pathway not previously taken into account was suggested.Our initial logical network recovered several experimental observations and produced predictions which were later experimentally validated34 we studied with our model the effect of niflumic acid (NFA) on the SASP by considering its action on the permeability of CaCC, HCN and CaKC. Though with slight differences, NFA affects these three channels in the μM range: CaCC40, CaKC42, and HCN45.In a more recent paperTwo scenarios emerged depending on the experimental controversy of whether the action of NFA on CaKC is inhibitory or activating. Taking into account the recently accumulated evidence regarding the activation of CaKCs by NFA, our model required the participation of more NFA-sensitive ionic channels in the SASP in order to recover the experimental results performed by ref. pHi-dependent Ca2+ channel in the Ca2+ signaling pathway. Prompted by the above, we added the pHi, voltage dependent CatSper channel to the network of ref. Ca2+ channels previously considered (HVA and LVA), by performing in silico knock-outs in our model network. The outcome that CatSper channel plays a leading role with regard to the amplitude of the Ca2+ fluctuations, lead us to a Model-III signaling pathway, where the only remaining Ca2+ channel is CatSper. With the three models at hand, we proceeded with a comparative study of their dynamics that lead us to the prediction that the CatSper channel participates in the S. purpuratus SASP and appears to be the principal generator of [Ca2+]i oscillations, while the other calcium channels considered in this study are probably secondary. Furthermore, our results prompted us to the design of pharmacological protocols to examine whether CatSper participates in the [Ca2+]i response induced by speract in S. purpuratus sperm. We confirmed by proteomics the presence of all CatSper subunits but Catsper3  previously considered in the SASP. The presence of this channel is further substantiated with the result that, with its addition, aforementioned shortcomings encountered in Model-I can be overcome.Our finding is that the contribution of the CatSper channel to the Ca2+ for Model-II averaged over 100,000 uniformly distributed random initial conditions determined from equation , a nonspecific PDE blocker, diminishes speract-dependent 48. The delay between the cGMP elevation and that of cAMP allows the KCNG channel to first open and hyperpolarize so that when cAMP begins to increase HCN can open and depolarize. Sperm population speract responses in the presence of IBMX have shown that membrane potential remains hyperpolarized indicating that cGMP activation of KCNG prevails over the cAMP induced depolarization caused by SpHCN49. IBMX wrecks the kinetic and spatial coordination between these two channels and [Ca2+]i increases but cannot decrease or oscillate. Since the CatSper channel requires a depolarization to activate even though a high hyperpolarization elevates pHi, CatSper will not open and a lower Ca2+ level will result.The results for Model-III  may be explained by considering that IBMX is a nonspecific phosphodiesterase inhibitor that allows accumulation of both cGMP and cAMP in all cell compartments. In a regular speract response cGMP first elevates in the flagella and with a delay cAMP then goes upCa2+ channel, a high hyperpolarization decreases the normal activity of CatSper, which in turn produces a lower Ca2+ level.Since the CatSper channel is a depolarization-activated Ca2+ channel; concomitant with the cGMP-dependent steps, which lead to a Ca2+ decrease, the absence of PDE also increases cAMP activity, which in turn stimulates a cAMP-modulated Ca2+ channel. So, in this case, we have a double Ca2+ regulation via cyclic nucleotides: an inhibitory (hyperpolarization-mediated) and an activator (cAMP-modulated Ca2+ channel opening). While for Model-II (panel C), the interplay between these opposite processes produces a higher Ca2+ level compared to the Model-III. The overall conclusion is that agreement with our results supports a dominant role displayed by CatSper with regard to the other Ca2+ channels: HVA and LVA.For the case of Model-I (panel B) with HVA, LVA and a cAMP-dependent Ca2+]i oscillations are expected to be pHi sensitive31. In Fig. i variations is indirectly modulated via a cAMPCC, the steady state oscillations in presence of HVA and LVA are basically unperturbed by the pH blockage. A similar situation is registered in panel B, for Model-II, now by the effect of CatSper instead of cAMPCC. For Model-III the direct connection to CatSper, in the absence of LVA and HVA causes a collapse in Ca2+ levels if the pH node is deleted. This is shown in panel C.[Ca2+]i oscillations, as well as an elevation on the average [Ca2+]i. This prompted S. purpuratus experiments, also reported in ref. Ca2+-dependent K+ channel Slo1, which showed an increase of the interval between successive Ca2+ oscillations, hence corroborating the prediction. However, a qualitative perception of the fluorescent intensity of the [Ca2+]i fluctuations was indicative of a lower average value than the WT case. Here, by considering the Ca2+ now averaged over 100,000 initial conditions we note in panel A of Fig. Ca2+ continues to be higher than the WT (in disagreement with the experiment) and the periodicity apparently is not substantially modified (again in contrast to experiment). To quantify the temporal behaviour of the Ca2+ dynamics, we performed a Fourier Spectrum Analysis.In ref. In a nutshell, peaks of the power spectra are indicative of the most important frequencies in these time series (Fourier modes). Since we have chosen the frequency representation in our Fourier analysis, the inverse of the frequency associated to each Fourier mode, corresponds to the most important periods of the calcium dynamics. These modes are dependent of the periodicity of the steady states reached by the whole network dynamics.Ca2+ concentration.Our Fourier Spectrum analysis, performed as in ref. 34, a study in terms of Model-I of the action of Niflumic Acid (NFA) on the flagellum membrane of S. purpuratus pointed either to the need of an additional Ca2+ channel such as the pH-dependent CatSper channel, not taken previously into account in its signaling pathway, or to the participation of a CaKC channel different from Slo1. With either choice, it was expected that the experimental determinations carried out in ref. in silico, namely: an increase in the average Ca2+ intracellular concentration, fluctuation amplitude, peak values and in the interval between successive oscillations. With Model-I, under the assumption that NFA blocks the HCN and CaCC channels and activates the CaKC channel, the experimental results are attained except for the latter one. In Fig. 17. Figure Ca2+ fluctuations averaged out over 100,000 initial conditions resulting from the dynamics of Model-III. Notice the presence of Fourier modes with periods higher than those encountered in the WT (i.e. in absence of NFA), coincident with the experimental determinations (red peaks at the left side of the figure).In our previous workCa2+]i fluctuations and their Fourier transforms  and absence (black) of NFA. The first red peak to the left corresponds to a period-12 Fourier mode, whilst the first black Fourier mode to the left of the wild type is indicative of a period-8 contribution. The link between the 12 steps red oscillation structure encompassed by the green line, that repeats itself periodically after 12 iterations, together with the period 12 Fourier mode, provides a manifestation of period elongation that goes beyond a guide to the eye. In general, Fourier analysis provides a means for exploring underlying regular components in the Ca2+ series, such as the coexistence of several initial condition dependent steady states. A more detailed study of the effect of NFA on the Ca2+ fluctuations, based on Fourier spectra can be found in ref. This assertion is reinforced by the joint analysis of the model-generated i increase, though both also block T-type voltage-dependent Ca2+ channels (LVA)51 (see discussion). These compounds were shown to inhibit the progesterone induced [Ca2+]i increases mediated by CatSper in human sperm52. Furthermore, during the course of these experiments, Seifert and colleagues32, showed all CatSper subunits are expressed in A. punctulata sperm and mibefradil was used to identify the participation of CatSper in chemotaxis. Figure Ca2+]i. These findings support the contention that CatSper channels have an important role in the response to speract and therefore in chemotaxis.The results obtained by the model network simulations motivated us to devise pharmacological experiments and examine the participation of CatSper channels in the SASP. Mibefradil and NNC-055-396 (NNC), two CatSper inhibitorsCa2+ Cav channels we had detected by immunocytochemistry in the flagella of sea urchin sperm8 and explore CatSper protein expression. Isolated flagella membranes53 from S. purpuratus were extracted with Triton X-114 to obtain a fraction enriched with integral membrane proteins54. The proteins in the Triton X-114 phase were resolved in either 10%  and the β, δ and γ subunits of this channel  based on the S. purpuratus CatSper1 predicted sequence (XP_011664201). The stained SDS-PAGE ; MW: 144 kDa)  with the model determinations is shown.The relevance of intracellular Ca2+ channel. It is worth noting that CatSper is present in the S. purpuratus genome57 and that here we corroborate that most of its reported protein subunits are expressed in sperm, as determined by proteomics. As anticipated, two CatSper blockers inhibit the speract induced [Ca2+]i increase, however this inhibition was partial. These results could suggest that another Ca2+ transport system participates. Recent observations in single S. purpuratus sperm showed that the speract-induced increase in flagellar pHi precedes and is independent of that of [Ca2+]i, therefore this alkalinization could directly activate a Ca2+ channel such as CatSper. However, an alkalinization induced by NH4Cl, which is larger than that caused by speract and that produces a membrane potential depolarization31, results in less Ca2+ influx, as compared with that triggered by speract. This finding would also be consistent with the required participation of another Ca2+ uptake mechanism in the speract response24.The table clearly indicates that best results are obtained with Model-III and that Model-II has some advantages over the Model-I. The detailed interpretations of this findings can be consulted in the results section. Our model hence predicts that assuming a 1-to-1 stoichiometry of all network elements, CatSper is indispensable and appears to be the main Ca2+]i oscillations, triggered by speract comes as an important theoretical prediction which requires alternate experimental confirmation. Besides the analysis here reported, credence of this result is further supported by previous successes of this approach in corroborating and predicting experiments34. A predictive capacity in the modeling of fundamental biological phenomena is encouraging and seldom encountered. In this respect a Systems biology approach with strong experimental backing is promising.Given the lack of specificity of CatSper blockers: NNC and mibefradil, neither our pharmacological experiments nor those presented in ref. The stoichiometry mentioned above is an important consideration. Additionally, it may be that a model in which the participation of the diverse channels is properly weighted may render a Model-II type network more appropriate. Further experimental work and alternative theoretical formulations such as continuum dynamics studies are required for a more thorough and conclusive analysis.Ca2+ channels, in Fig. Ca2+ curve with a faster decay, thus influencing the dynamical behaviour of the system under that perturbation, but maintaining almost unaltered the average calcium concentration as well as the elevation rate. Since phenomena like chemotaxis and chemokinesis are strongly related to the temporal synchronization of the signaling pathway and the molecular machinery with spatio-temporal gradients of egg-derived molecules15, we suggest that the the presence of those channels could intervene in the fine tunning necessary for a correct movement of the sperm in its searching for the egg.With regard to the other The construction of the regulatory functions mentioned in the SASP network model section is better understood by means of an example regarding the regulation of cyclic GMP (cGMP). This molecule is a well known second messenger in almost every cell type. It is synthesized by guanylate cyclase (GC) and degraded by the phosphodiesterases (PDE). In this case, cGMP, GC and PDE take two values: 0 and 1. For the construction of its regulatory function it is necessary to take into account all possible combinations of nodes linked to it. At time t, if PDE is active (state 1), cGMP cannot be synthesized, even if GC is active. This is because PDE is a strong inhibitor. When PDE state is 0, if GC or cGMP are in state 1, cGMP will be on at time (t + 1). This table has positive autoregulation of cGMP. Table This construction was applied to all nodes in the system. It is important to mention that several criteria can intervene in the construction of a regulatory function and in order for it to be meaningful an in depth biological knowledge is essential.34, we have assumed this occurs in sea urchin sperm. We simulated an activation effect by forcing them to take the value of 1 during a certain period, for example 3 steps, and then releasing them in the fourth step. Actually, the network dynamics results are not strongly dependent on the specific values of the above steps. For the Fig. For node elimination in the network dynamics, a zero value was imposed on it during the whole dynamics independently of its regulators. In the particular case of CaKC and CatSper, though there are evidences in mouse sperm suggesting CatSper is stimulated by low NFA concentrationCa2+]i fluctuations by averaging the value of the [Ca2+] node for each step in the dynamics over 105 different initial conditions of the entire network  once steady state periodic oscillations were reached. Since our nodes take a finite number of values and the network is finite, periodic behaviours are always attained. Though the initial conditions are chosen according to a uniform distribution, oscillatory phases are determined by the distribution of the time of arrival to the periodic steady state. In our study we compared averaged intracellular Ca2+ ion concentration fluctuations determined from the model dynamics under consideration with experimentally measured [Ca2+]i levels determined as averaged values taken over flagellum length. With this procedure we obtain a finer resolution for the Ca2+ concentration, more adequate for comparison with experimental measurements, as well as more appropriate for the calculation of systemic features.We determined model generated speract-triggered i oscillations in individual sea urchin sperm and in populations. Controls have been done showing that these fluctuations are not due to Fluo-4 pH-dependent fluorescent changes.Sperm fluorescence measurements were done as describedp < 0.05.Fluorescence records were acquired with 4.2 Global Works OLIS software and processed with Microsoft Excel. The values given in the bar plots are the mean ± s.e.m and n = independent experiments. For statistical evaluations we used the Student’s t-test. Differences were considered statistically significant when Supplementary InformationDataset S1Dataset S2Dataset S3


Astrocytes are fundamental players in the regulation of synaptic transmission and plasticity. They display unique morphological and phenotypical features that allow to monitor and to dynamically respond to changes. One of the hallmarks of the astrocytic response is the generation of calcium elevations, which further affect downstream cellular processes. Technical advances in the field have allowed to spatially and to temporally quantify and qualify these elevations. However, the impact on brain function remains poorly understood. In this review, we discuss evidences of the functional impact of heterogeneous astrocytic calcium events in several brain regions, and their consequences in synapses, circuits, and behavior. These might appear as “global” and/or “focal” responses within the astrocytic complex morphology. It is yet unclear if global Ca2+ elevations are more representative of an integrative response or if they are the result of a linear summation of Ca2+ fluctuations or inositol 1,4,5-trisphosphate (IP3) levels; nevertheless, these have been used for over two decades as a readout of astrocytic function  triggers the IP3 signaling cascade and results in robust intracellular Ca2+ elevations, mainly via the IP3 receptor type 2 activation (IP3R2) (B receptor (Gi-coupled GPCRs) activation was also shown to trigger intracellular Ca2+ elevations in astrocytes   ; for rev (IP3R2) . Curioustrocytes . Moreovetrocytes . The spotuations , 2013, a+ levels . Additioy (SOCE) . More re2+ signaling is steadily growing, its functional consequences to the surrounding cellular circuits remain poorly understood. Therefore, this review will focus on the available data showing functional outputs of astrocytic Ca2+ elevations. The reader may find useful reviews and discussions elsewhere on the origin and features of astrocyte Ca2+ elevations . Different levels of neuronal activity appear to scale astrocytic Ca2+ levels, leading to orchestrated (multi)synaptic responses  of hippocampal CA1 pyramidal cells  in vicinal pyramidal neurons. Furthermore, endocannabinoid-triggered elevation of astrocytic Ca2+ levels contributes to heteroneuronal LTP  Caefficacy . Similarsynapses . The Ca2ia GAT-3 . In addiia GAT-3 .2+ elevations, namely by using Ca2+ chelators or genetic IP3R2 deletion. This may explain the current bias toward effects of global (soma and main processes) Ca2+ signals rather than that of focal events (restricted to cellular microdomains). Nevertheless, it is now clear that global and focal signals coexist in specific spatio-temporal maps. Despite the 10- to 100-fold scale that distinguishes global and focal signals, both have been shown to precede transmitter release and modulate different forms of synaptic transmission . First, the activation of astrocytes appears to represent a novel integration mechanism. Astrocyte Ca2+ elevations not only occur upon activation through various excitatory transmitters , but can also be triggered by the inhibitory transmitter GABA. This means that, independently of the nature of the transmitter, astrocytes will be excited/activated , suggesting they integrate some brain circuits as a redundant layer that reads excitatory and inhibitory neuronal inputs similarly. This concept may add up to the mechanisms of coincidence detection proposed a decade ago by 2+ elevation, astrocytes release gliotransmitters that ultimately cause either neuronal excitation  or inhibition . Curiously, the available literature indicates that astrocytes provide, in most cases, an excitatory output. Nevertheless, astrocytes also release ATP, which is readily degraded to adenosine and in turn activates A1 receptor leading to synaptic inhibition  also have an expected impact on brain circuits.The difficulty to measure and/or manipulate intracellular astrocytic Ca2+ elevations in vivo regulate extracellular glutamate levels, which consequently triggers a slow neuronal rhythm in the brain. This event is characterized by synchronized neuronal firing across different behavioral states, and points to an important regulatory role of astrocytes in cortical circuits -dependent plasticity in the somatosensory cortex in vivo. This process is dependent on IP3R2 signaling and extracellular D-serine  triggering intracellular Ca2+ elevations via chemogenetic activation of GPCR signaling, and (2) inhibiting IP3 signaling by deletion of IP3 receptors or by buffering IP3.The study of rodent models that display altered astrocyte function indicated that astrocytes play important roles in the production of behavior outputs in different dimensions  -positive cells , does not appear to influence spatial memory , these two studies provided only modest evidence to support it. We believe that further studies using more specific tools to modulate astrocyte Ca2+ will reveal additional links to cognition. Indeed, we recently showed that spatial learning and memory rely on astrocyte exocytosis, which is a Ca2+-dependent mechanism, and therefore might be dependent on astrocyte integration of surrounding activity  phase and the frequency of REM periods , or by the promotor used to drive astrocyte specificity (GFAP vs. GLT1) that could result in differential regional expression. Moreover, there are at least five different IP3R2 KO strains with different genetic backgrounds  and influence circuit outputs (sensory plasticity and network synchronization). However, the behavior readouts of rodents whose global astrocytic Ca2+ was manipulated remain inconsistent.Intracellular Ca2+ signals exhibit critical spatio-temporal features that need to be fully understood to allow for the careful assessment of their functional implications. Among them: spontaneous vs. evoked events; fast vs. slow elevations; synchronous vs. asynchronous; global (soma and main processes) vs. focal (microdomain); single vs. multiple astrocyte; local vs. across brain regions; IP3-dependent vs. IP3-independent mechanisms. The complementary application of existing tools, as well as the development of accurate models to tackle its unique and heterogeneous features, will provide us with novel insights on its actions and consequences.Astrocytic CaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.The necessity of extracellular CaX. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.Using the appearance of cleavage furrows as an indicator of embryonic development, we found that 2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu.Taken together, these data are consistent with requirement of extracellular Ca Fertilization evokes increased intracellular Ca2+ in the zygote, and this Ca2+ is essential for egg activation and the earliest events of embryonic development in all sexually reproducing species studied thus far [2+ regulates various cellular events in both gametes [2+ during fertilization is controversial. For example, entry of extracellular Ca2+ into sperm signals processes required for fertilization such as the acrosome reaction [2+ is required for robust motility in X. laevis sperm [2+ [2+ is unessential for fertilization in X. laevis [Fertilization represents the union of two distinct cell types: the sperm and the egg. Although a substantial amount of work on various species has already been done, many of the signaling pathways and molecular events required for fertilization are unknown. Cathus far , 2. More gametes –7. By cois sperm . Howeverperm [2+ . Moreove. laevis –13.2+ is required for fertilization and normal embryonic development in X. laevis eggs. The Ca2+ chelators BAPTA ethane-N,N,N′,N′-tetraacetic acid) and EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) inhibited embryonic development in a concentration-response manner, with almost no embryonic development evident in eggs inseminated in chelator concentrations of 3 mM or higher. Incubation of eggs and zygotes in BAPTA at various times before or after insemination revealed that extracellular Ca2+ is necessary within the first 30 minutes of X. laevis insemination. We also found that BAPTA-treated sperm do not penetrate the eggs’ vitelline envelope, consistent with the requirement of extracellular Ca2+ for X. laevis sperm to undergo the acrosome reaction [2+. Taken together, these results indicate that extracellular Ca2+ is crucial for fertilization in X. laevis and that the jelly coat serves as a readily available source of diffusible Ca2+.Here we report that extracellular Careaction . FinallyAll chemicals, unless noted, were purchased from Thermo Fisher Scientific .BAPTA  and EGTA were prepared as 10 mM or 50 mM stock solutions (adjusted to pH of 7.8 with NaOH) in deionized water and stored at 4°C until use.2, 1 mM MgCl2, and 5 mM HEPES, pH 7.8) [2 and MgCl2. Fertilization solutions were prepared by diluting either DVF or MR with purified water to the desired concentration, either 33% MR (MR/3) or 33% DVF (DVF/3) [All solutions were made in reverse osmosis treated and Elga purified water . Most gamete preparations and experiments were performed using a version of filter sterilized Modified Ringer’s (MR) solution  . Experim (DVF/3) , unless X. laevis frogs were obtained commercially  and housed at 18°C on a 12 hour/12 hour light/dark cycle. Ovulation was induced in sexually mature X. laevis females with injection of 1000 IU of human chorionic gonadotropin (hCG) into the dorsal lymph sac. Following hCG injection, individuals were housed overnight for 14–16 hours at 14°C. Females began ovulating 0–2 hours after moving to room temperature. Eggs were collected on dry plastic petri dishes and were used within 10 minutes of collection.All animal protocols were conducted using accepted standards of humane animal care, approved by the Animal Care and Use Committee at the University of Pittsburgh. Adult X. laevis males that were euthanized by immersion in 3.6 g/L tricaine pH 7.4, for 30 minutes. Dissected testes were stored at 4°C in MR for use the day of dissection or in L-15 media (Gibco 11415–064) for use up to 5 days later. To create a sperm suspension, 1/10 of a testis was minced in DVF. Eggs were inseminated by pipetting the sperm into the petri dish above the egg; the volume of added sperm solution never exceeded 1% of the total fertilization solution.Testes were collected from sexually mature 2+ or chelators and then assessed for development. Development was assessed based on the appearance of cleavage furrows, which were typically apparent approximately 90 minutes after sperm addition. Inseminated eggs that did not develop cleavage furrows were scored as undivided. Cleaved embryos were scored as developed at the two-, four-, and eight-cell stages. Each experiment was repeated at least three times, and the depicted error bars represent the standard error of the mean (SEM). Data were analyzed with Igor  and statistical analyses were performed in Excel (Microsoft).Approximately 15–30 eggs per treatment were inseminated in DVF/3 with or without Ca2. These plots were then fit with a sigmoidal function (equation 1):Y0 represents the minimum response, Y∞ is the maximum response, xhalf represents the half maximal response, and the rate is the slope of the curve.Concentration-response relationships were calculated by plotting the averaged percentage of developed embryos per experiment versus added concentrations of chelator or CaClTransfer assays were performed at various time points relative to sperm addition. For these assays, eggs were inseminated in one solution, washed twice, and then transferred into a different solution 5 or 30 minutes after sperm addition. For 5 minute transfer assays, sperm were minced in DVF/3. Eggs and embryos were incubated in petri dishes coated with 1% agarose dissolved in a DVF/3 solution, and plastic transfer pipettes were used to move inseminated eggs between treatments.In order to assess whether sperm penetrated the vitelline envelope of BAPTA-treated eggs, eggs were inseminated in 0 or 3 mM BAPTA with 20 μg/ml Hoechst. 20 minutes after insemination, egg jelly was removed with 45 mM β-mercaptoethanol in basic DVF/3 (pH 8.5), washed twice with acidified DVF/3 (pH 6.5), then transferred into the starting solution, DVF/3 with 0 or 3 mM BAPTA . Dejelli2+ content within the jelly coats surrounding X. laevis eggs, freshly ovulated eggs were incubated in DVF/3, and then this DVF/3 was assayed for enriched Ca2+ content. Accordingly, approximately 70–150 eggs were incubated in 4 or 5 ml of DVF/3 for each trial. The DVF/3 was collected and changed every 10–30 minutes for three hours to collect and deplete readily available Ca2+ content of the jelly. The Ca2+ content of each 4 or 5 ml DVF/3 application was quantified with fura-2 photometry.To estimate the readily available Ca2+ bound (343 nm) and Ca2+ free (370 nm) fura-2 spectra [min (2.4), Rmax (6.8), and K* (28 μM), obtained from DVF/3 solutions supplemented with known amounts of CaCl2 ranging from 10 nM– 30 mM. The sum of total Ca2+ content from all washes made from a single trial was calculated and is reported as the average ± SEM.Fura-2 pentasodium salt (Alfa Aeser) was dispensed from a 100 μM stock in water for a final concentration of 100 nM in each DVF/3 aliquot. Fluorescence intensity measurements were recorded in a 1 mm quartz cuvette, in a Fluorolog 3 spectrophotometer with FluorEssence software . Fura-2 samples were excited with 250–450 nm light, and emission was recorded at 510 nm with 3 nm slit widths. The raw photometric signals were corrected for by subtracting the fura-2 free background, collected prior to each series of measurements. The ratios were measured with 343 and 370 nm excitation light, representing the acquired excitation peaks of the Ca spectra . The rat spectra  with equ2+ in the jelly coat surrounding the eggs, the total Ca2+ content measured by fura-2 photometry was divided by the volume of solution in each wash, the number of eggs per incubation, and the average volume of the jelly coat. Jelly coat volumes were quantified from images acquired with a 10X objective on an Edmund Optics stereomicroscope, which was fitted with a pixiLINK digital camera and the μScope Essential x64 software . Measurements were made from these acquired images by determining the diameter of the eggs and their surrounding jelly coat in Adobe Illustrator .To determine the concentration of diffusible Ca2+ during X. laevis fertilization and early embryonic development, we inseminated eggs in solutions with and without the addition of the divalent cations Ca2+ and Mg2+. The incidence of cleavage furrow development  was not significantly different between these two treatments with 97 ± 1% development in MR/3, compared to 93 ± 4% in DVF/3 (X2 (1) = 1.96, P = 0.07, Pearson’s chi-squared test) (top), and eggs that did not develop cleavage furrows were scored as undivided (middle).To examine the requirement of extracellular Caed test) . Develop2+ during fertilization and early embryonic development, we inseminated X. laevis eggs in DVF/3 that included varying concentrations of the Ca2+ chelator BAPTA ranging from 10 μM to 5 mM. We found that only 12 ± 4% of eggs that were inseminated in 1 mM BAPTA exhibited embryonic development , and that no normal development occurred in eggs inseminated in 3 mM or 5 mM BAPTA  (50) of 519 ± 76 μM .To investigate the absolute requirement of extracellular Ca trials) . By cont trials) . Fitting2+ rather than a non-specific effect or lack of Mg2+, eggs were inseminated in the presence of 1 mM BAPTA with varying concentrations of added CaCl2 ranging from 10 μM to 5 mM. The averaged percentage of development was plotted against the total added CaCl2 concentration (2+ concentration (EC50) of 420 ± 40 μM . Together these data demonstrate that extracellular Ca2+ rescues the effects of BAPTA treatment.To verify that the lack of embryonic development seen in eggs inseminated in BAPTA was due to the absence of extracellular Cantration . We agai2+ chelator, EGTA, also precluded embryonic development. To do so, we inseminated X. laevis eggs in DVF/3 with various concentrations of EGTA ranging from 3 μM to 3 mM. Similar to BAPTA, insemination in EGTA reduced the incidence of cleavage furrow development in a concentration-dependent manner with an average IC50 of 178 ± 25 μM  . Interestingly, 96 ± 2% the embryos transferred from 0 to 3 mM BAPTA developed faint cleavage furrows with blastomeres that appeared less round, a phenotype not seen under typical fertilization conditions (bottom vs. middle).Many imperative events in embryonic development occur in the 90 minutes that it typically takes for cleavage furrows to appear. Determining which developmental events require extracellular Ca trials) . These r2+ is the acrosome reaction [X. laevis, this particular matrix is called the vitelline envelope. Following fertilization, the egg releases its cortical granules into the perivitelline space, and enzymes contained within these granules transform the vitelline envelope into the fertilization envelope [Eggs inseminated in the presence of 3 mM BAPTA or higher failed to develop cleavage furrows in all experimental conditions examined thus far. However, these assays have not distinguished between inhibition of events occurring before or immediately after fertilization. One fertilization event thought to require extracellular Careaction . The acrreaction ; and in envelope . Whereasenvelope , consequ2+ chelator. To test this experimentally, we imaged the perivitelline space to assay for the presence of Hoechst-stained sperm, in eggs and embryos inseminated with or without 3 mM BAPTA. To facilitate imaging, the jelly surrounding these eggs was removed using a reducing solution, which leaves the vitelline and fertilization envelopes intact [top), and in 55 out of 56 eggs inseminated in 3 mM BAPTA with 3 mM Ca2+ in 4 experimental trials (bottom). By contrast, we did not identify any sperm near eggs inseminated in 3 mM BAPTA  (middle). These data suggest that sperm were unable to penetrate the vitelline envelope in the presence of BAPTA.We reasoned that if high BAPTA concentrations prevented the acrosome reaction under our experimental conditions, then sperm would only penetrate the vitelline envelope of eggs inseminated without the Cas intact . The Hoel trials bottom.  trials) . middle.2+ concentrations. We predicted that fertilization should be possible following the five-minute transfer as sperm should be viable and fertilization competent within 10 minutes following extraction from the testes [2+ is not required for sperm to penetrate the jelly surrounding the egg, then sperm should enter the jelly coat of inseminated eggs regardless of whether or not BAPTA is present. Only sperm within the jelly coat should be transferred to the new solution. We found that embryos inseminated in 3 mM BAPTA and 5 minutes later transferred to a Ca2+-containing MR/3 solution, developed cleavage furrows   . Further2+ is required for fertilization. However, it was not yet clear why DVF/3, which should have a low (≤ tens of nM) Ca2+ content, supported fertilization and embryonic development, yet interference with embryonic development required high micromolar concentrations of BAPTA or EGTA. We hypothesized that the jelly surrounding X. laevis eggs may include a readily available store of Ca2+ ions, and that high BAPTA concentrations deplete this Ca2+ source. Another set of conditions was added to the above transfer assay to test this hypothesis. Specifically, eggs were inseminated in 3 mM BAPTA, a condition that based on our experimental observations should substantially reduce the putative Ca2+ store. Five minutes following sperm addition, eggs were washed in and transferred to DVF/3, a condition that should not replenish the Ca2+. Indeed, we found little embryonic development, 12 out of 85 eggs inseminated in 3 mM BAPTA and transferred to DVF/3 five minutes following sperm addition . All 12 embryos developed faint cleavage furrows (bottom). Together these results suggest that the BAPTA is removing Ca2+ from the extracellular matrix, and that only MR/3, and not DVF/3, replenishes this Ca2+ to support fertilization.The data collected thus far indicated that at least nominal extracellular Ca furrows bottom. 2+. To do so, we estimated the diffusible Ca2+ content from the jelly using fura-2 photometry of DVF/3 following serial incubations of X. laevis eggs. In four experimental trials, an average of 17.5 ± 4.7 nmol of Ca2+ diffused from each jellied egg into the surrounding DVF/3 . Based on an averaged diameter of jellied eggs of 2.0 ± 0.2 mm, and an averaged diameter of 1.4 ± 0.02 mm for the egg alone, and the assumption that the egg and surrounding jelly coat are spherical, we estimate that the averaged jelly volume to be 2.8 μl (N = 11 jellied eggs from 4 frogs). Accordingly, we estimate that the averaged concentration of freely-diffusing Ca2+ in the jelly coat is 6.3 ± 1.7 mM.We next explored the hypothesis that the jelly coat surrounding the egg is enriched with readily available Ca2+ is unessential for fertilization and embryonic development in X. laevis [X. laevis. BAPTA and EGTA are both chelators that share a high selectivity (>105) for Ca2+ over Mg2+, and each has empirically been shown to bind only a single Ca2+ ion [2+ and Mg2+. However, the ability of Ca2+ to recover fertilization in high BAPTA concentrations  for the BAPTA-Ca2+ interaction at pH 7.8 is 110 nM [50 for the BAPTA inhibition of embryonic development is three orders of magnitude higher at 519 ± 76 μM , and the sperm-specific Ca2+ channel CatSper. The CaSR has been hypothesized to facilitate the HCO3- signaled capacitation in mammalian sperm [3- signaled capacitation events including increased sperm motility and tyrosine phosphorylation [3- signaled capacitation requires extracellular Ca2+ [2+ may also be required for entry into the sperm-specific channel CatSper. In mammals, Ca2+ entry via CatSper is required for a swimming pattern termed hyperactivation [X. laevis sperm for fertilization as it does for mammalian sperm.Extracellular Caan sperm . Capacitan sperm . A role an sperm  and stalan sperm . Second,rylation , 31. Thilar Ca2+ . Similartivation , an essetivation . CatSpertivation . To date2+ chelators, they also bind to other transition metals including Zn2+ [3+ [2+ is also disrupting fertilization and early embryonic development in X. laevis. Notably, Zn2+ has a documented requirement in mouse oocyte maturation into fertilization-competent eggs [2+ are released from the embryo into the extracellular space in the same timeframe of the cortical granule reactions in mammalian eggs [2+ plays in X. laevis fertilization is not yet understood but it may prove to be essential. Transition metals other than Ca2+ are known to bind proteins required for fertilization, such as the sperm protein matrix metalloproteinase-2 (MMP-2) [X. laevis eggs, and is required for fertilization [X. laevis, we believe that the ability of Ca2+ to overcome the blockade imposed by high concentrations of BAPTA suggest that Ca2+ is necessary for these events (Although BAPTA and EGTA are widely recognized as Caing Zn2+  and Fe3+Zn2+ [3+ . It is pent eggs . Large qian eggs . The rol (MMP-2) . MMP-2 ilization . As a mee events .2+ for fertilization and embryonic development in X. laevis. Here we present several pieces of data demonstrating for the first time that Ca2+ is absolutely required for fertilization and embryonic development in X. laevis. Furthermore, our data indicates that the jelly coat surrounding X. laevis eggs includes a store of readily available Ca2+ ions. Although the exact ionic composition of X. laevis jelly is yet to be determined, we believe the high Ca2+ content of the jelly coat may serve as a protective environment to optimize fertilization conditions in changing external environments.We sought to resolve the controversial requirement of extracellular Ca


Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.4Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca2+ concentration ([Ca2+]i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca2+]i in astrocytes and ECs.Following acute application and removal of NH4Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH4Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH4Cl, a transient, concentration-dependent rise in [Ca2+]i in astrocytes was observed. This was due to the release of Ca2+ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca2+-free bathing solution. The removal of NH4Cl also led to a transient concentration-dependent rise in [Ca2+]i that resulted from Ca2+ release from cytoplasmic proteins, since removing Ca2+ from the bathing solution and emptying intracellular Ca2+ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH4Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH4Cl, and EC shrinkage was demonstrated after the acute removal of NH4Cl.Exposure of astrocytes and ECs to 1 – 20 mM NHThis study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE. Hepatic encephalopathy (HE) comprises a broad spectrum of neurological and psychiatric dysfunctions seen in patients with liver disease. It can reflect diverse conditions, such as a reversible metabolic encephalopathy, brain atrophy, brain edema or a combination of them. Prevalent hypotheses suggest that a number of metabolic factors, present at the same time, are responsible for the development of HE. Neurotoxins, alteration of the blood-brain barrier (BBB), impairment of neurotransmission, altered brain energy metabolism and systemic response to infections and neuroinflammation are all factors playing a role in the pathogenesis of HE –3.Ammonia is the most studied and well-described neurotoxin that precipitates HE –5, since2+]i [2+]i may also play an important role modulating brain microcirculation i , 15, 16 2+]i . Changesculation . Howevered cells .4Cl concentrations. Hyperammonemia did not produce brain edema in experimental animals in vivo i in astrocytes following the application of ammonia have been studied [2+]i changes after the acute removal of ammonia in order to give a more complete insight into the mechanisms of HE treatment. Additionally, the role of changes in EC morphology in the development of brain edema has been evaluated by analyzing changes in pH, [Ca2+]i and volume in ECs following the addition and subsequent removal of ammonia.Changes of pH and i. During experiments SBS was exchanged as appropriate for a solution with a different concentration of ammonia which, after 10 min, was again changed for SBS i, astrocytes were labeled with Fura-2/AM at 4 μM. The protocol was similar to that for [H+]i measurement, except that excitation wavelengths of 340 nm (F340) and 380 nm (F380) and a filter passing wavelengths higher than 510 nm were used [340/F380) was used to determine [Ca2+]i.To determine i were further analyzed with the ImageJ  computer software for measuring cell areas. The contrast of the images was enhanced to better determine cell borders. Each visible cell was then outlined at the beginning of the experiment, before and after each solution exchange and at the end of the experiment. Changes of areas were then calculated.Images of those cells acquired during the experiments while measuring i were observed using the calcium indicator Fura-2/AM. Given that a large proportion of the intracellular Ca2+ is bound to cytoplasmic proteins, an increase in intracellular pH caused by the addition of NH4Cl should result in a decrease of [Ca2+]i. The reduction in [H+]i results in the release of H+ from cytoplasmic proteins; thus free Ca2+ ions fill up the freed protein-binding sites [i should lead to a fall of [Ca2+]i. However our results, as well as those of previous studies [2+]i rises after acute alkalization of cytoplasm by NH4Cl  increase of F340/F380 and the addition of 5 mM and 20 mM NH4Cl resulted in 28.1 % ± 16.6 %  and 59.5 % ± 17.2 %  increases respectively , suggesting that intracellular Ca2+ stores must be responsible for the rapid increase in [Ca2+]i following exposure to NH4Cl. In a Ca2+-free bathing solution, exposure to 20 mM NH4Cl resulted in an increase of F340/F380 of 54.6 % ± 19.6 % .Since the rise in i following the addition of NH4Cl resulted in release of H+ from cytoplasmic proteins. The newly freed protein-binding sites were filled by intracellular Ca2+, resulting in a reduction in [Ca2+]i.The suggestion that Care added  to the C2+ from intracellular stores demonstrated after alkalinization of astrocytes by NH4Cl is consistent with reported results i. Even after depleting intracellular stores with thapsigargin and ATP, the removal of NH4Cl led to an increase in F340/F380 of 79.3 % ± 27.6 % . The [Ca2+]i rise is probably a result of the release of Ca2+ from cytoplasmic proteins.A transient rise in F+ and Ca2+ concentrations in astrocytes that occurred on substituting SBS with 20 mM NH4Cl, then resubstituting the bathing solution back with SBS, changes in cell volume following these acute events were investigated. Volume changes immediately and approximately 10 min after acute exchange of the bathing solutions were determined by confocal microscopy. However, no significant changes in astrocyte volume during the experiment were detected, which is consistent with other studies, in which a progressive increase of the cell volume of astrocytes exposed to NH4Cl was observed only after 1–3 days i. After the addition of NH4Cl, a small increase in F340/F380  was observed but, after its removal, a rise in F340/F380  similar to that observed in ECs in Ca2+-free solution without intracellular Ca2+ stores being depleted (p = 0.092) was measured.Intracellular Caf 100 μM  before m4Cl to ECs triggers a transient increase in [Ca2+]i that is attenuated in ECs in Ca2+-free bathing solution and almost abolished in ECs after the depletion of intracellular Ca2+ stores in the Ca2+-free bathing solution. However, acute removal of NH4Cl from the extracellular solution provoked a transient rise in [Ca2+]i which could not be abolished, either by substituting the SBS with Ca2+-free bathing solution or by further depleting intracellular Ca2+ stores, suggesting that the increase in [Ca2+]i is due to a release of Ca2+ from cytoplasmic proteins.Our results suggest that the addition of NH4Cl i were measured. Exposure of EC to different solutions was relatively short, and it is not possible to exclude further volume changes of ECs that might have appeared after a longer exposure to NH4Cl.Volume changes of ECs were measured after the administration and subsequent removal of NH4Cl , and returning to SBS resulted in a markedly smaller cell shrinkage of 11.0 % ± 13.0 %   was recorded after changing SBS for 20 mM NH4Cl  after substituting 20 mM NH4Cl with SBS  Fig. . While o4Cl Fig.  and an aSBS Fig. . The resculation , but ourmmonemia . This ismmonemia . It is pmmonemia .Fig. 7Ch4Cl elicits a transient rise of intracellular Ca2+ concentration in astrocytes and ECs. This is consistent with the results of previous studies i is shown to be due to release of Ca2+ from cytoplasmic proteins. The present study also shows that the addition and removal of NH4Cl results in an increase and decrease respectively of EC volume. Such effects were, however, absent in astrocytes. Moreover, the removal of NH4Cl, which is the basis of modern treatment of HE, resulted in a decrease in EC volume despite an accompanying transient increase in [Ca2+]i. Further studies are needed to determine the connection between these events.Acute addition of NH studies . The acu2+]i, intracellular calcium concentration; ECs, endothelial cells; [H+]i, intracellular H+ concentration; HA, hyperammonemia; HE, hepatic encephalopathy; pHi, intracellular pH; SBS – standard bathing solution.ATP, adenosine-5’-triphosphate; BBB, blood–brain barrier; [Ca