scholarly journals Histones and basic polypeptides activate Ca2+/cation influx in various cell types

1998 ◽  
Vol 331 (2) ◽  
pp. 623-630 ◽  
Author(s):  
Alessandra GAMBERUCCI ◽  
Rosella FULCERI ◽  
Paola MARCOLONGO ◽  
William F. PRALONG ◽  
Angelo BENEDETTI
Keyword(s):  
Rat Hepatocytes ◽  
Cell Types ◽  
Membrane Depolarization ◽  
Histone H2a ◽  
Specific Manner ◽  
A Cell ◽  
Cell Depolarization ◽  
Influx Pathways ◽  
Basic Polypeptides ◽  
Ca2 Channels

Histone H2A (1–10 µg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr > 4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.

scholarly journals The complement of desmosomal plaque proteins in different cell types.

1985 ◽  
Vol 101 (4) ◽  
pp. 1442-1454 ◽  
Author(s):  
P Cowin ◽  
H P Kapprell ◽  
W W Franke
Keyword(s):  
Guinea Pig ◽  
Cell Types ◽  
Cardiac Cells ◽  
Myocardial Tissue ◽  
Cell Type ◽  
Wide Range ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific ◽  
Different Cell Types

Desmosomal plaque proteins have been identified in immunoblotting and immunolocalization experiments on a wide range of cell types from several species, using a panel of monoclonal murine antibodies to desmoplakins I and II and a guinea pig antiserum to desmosomal band 5 protein. Specifically, we have taken advantage of the fact that certain antibodies react with both desmoplakins I and II, whereas others react only with desmoplakin I, indicating that desmoplakin I contains unique regions not present on the closely related desmoplakin II. While some of these antibodies recognize epitopes conserved between chick and man, others display a narrow species specificity. The results show that proteins whose size, charge, and biochemical behavior are very similar to those of desmoplakin I and band 5 protein of cow snout epidermis are present in all desmosomes examined. These include examples of simple and pseudostratified epithelia and myocardial tissue, in addition to those of stratified epithelia. In contrast, in immunoblotting experiments, we have detected desmoplakin II only among cells of stratified and pseudostratified epithelial tissues. This suggests that the desmosomal plaque structure varies in its complement of polypeptides in a cell-type specific manner. We conclude that the obligatory desmosomal plaque proteins, desmoplakin I and band 5 protein, are expressed in a coordinate fashion but independently from other differentiation programs of expression such as those specific for either epithelial or cardiac cells.

scholarly journals Characteristics and Functions of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionate Receptors Expressed in Mouse Pancreatic α-Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1541-1550 ◽  
Author(s):  
Jung-Hwa Cho ◽  
Liangyi Chen ◽  
Mean-Hwan Kim ◽  
Robert H. Chow ◽  
Bertil Hille ◽  
...  
Keyword(s):  
Glutamate Receptor ◽  
Ampa Receptors ◽  
Islet Cells ◽  
Hormone Secretion ◽  
Cell Types ◽  
Membrane Depolarization ◽  
Voltage Gated ◽  
Mouse Pancreatic Islets ◽  
Inward Currents ◽  
Ca2 Channels

Pancreatic islet cells use neurotransmitters such as l-glutamate to regulate hormone secretion. We determined which cell types in mouse pancreatic islets express ionotropic glutamate receptor channels (iGluRs) and describe the detailed biophysical properties and physiological roles of these receptors. Currents through iGluRs and the resulting membrane depolarization were measured with patch-clamp methods. Ca2+ influx through voltage-gated Ca2+ channels and Ca2+-evoked exocytosis were detected by Ca2+ imaging and carbon-fiber microamperometry. Whereas iGluR2 glutamate receptor immunoreactivity was detected using specific antibodies in immunocytochemically identified mouse α- and β-cells, functional iGluRs were detected only in the α-cells. Fast application of l-glutamate to cells elicited rapidly activating and desensitizing inward currents at −60 mV. By functional criteria, the currents were identified as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. They were activated and desensitized by AMPA, and were activated only weakly by kainate. The desensitization by AMPA was inhibited by cyclothiazide, and the currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Islet iGluRs showed nonselective cation permeability with a low Ca2+ permeability (PCa/PNa = 0.16). Activation of the AMPA receptors induced a sequence of cellular actions in α-cells: 1) depolarization of the membrane by 27 ± 3 mV, 2) rise in intracellular Ca2+ mainly mediated by voltage-gated Ca2+ channels activated during the membrane depolarization, and 3) increase of exocytosis by the Ca2+ rise. In conclusion, iGluRs expressed in mouse α-cells resemble the low Ca2+-permeable AMPA receptor in brain and can stimulate exocytosis.

scholarly journals Phospholipase C-γ1 is required for the activation of store-operated Ca2+ channels in liver cells

2007 ◽  
Vol 405 (2) ◽  
pp. 269-276 ◽  
Author(s):  
Tom Litjens ◽  
Than Nguyen ◽  
Joel Castro ◽  
Edoardo C. Aromataris ◽  
Lynette Jones ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Maximal Effect ◽  
Experimental Conditions ◽  
Induced Changes ◽  
Molecular Components ◽  
Interfering Rna ◽  
Ca2 Channels

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-γ (phospholipase C-γ) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-γ-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-γ has not been defined clearly. The aim of the present study was to determine if PLC-γ is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-γ1 caused a reduction (by approx. 70%) in the PLC-γ1 protein expression, with maximal effect at 72–96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the ISOC (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP3 or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the ISOC amplitude. Immunoprecipitation of PLC-γ1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-γ1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

scholarly journals Arachidonic acid inhibits the store-operated Ca2+ current in rat liver cells

2005 ◽  
Vol 385 (2) ◽  
pp. 551-556 ◽  
Author(s):  
Grigori Y. RYCHKOV ◽  
Tom LITJENS ◽  
Michael L. ROBERTS ◽  
Greg J. BARRITT
Keyword(s):  
Arachidonic Acid ◽  
Rat Liver ◽  
Membrane Conductance ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Receptor Activation ◽  
Liver Cells ◽  
H4iie Cells ◽  
Rat Liver Cells ◽  
Ca2 Channels

Vasopressin and other phospholipase-C-coupled hormones induce oscillations (waves) of [Ca2+]cyt (cytoplasmic Ca2+ concentration) in liver cells. Maintenance of these oscillations requires replenishment of Ca2+ in intracellular stores through Ca2+ inflow across the plasma membrane. While this may be achieved by SOCs (store-operated Ca2+ channels), some studies in other cell types indicate that it is dependent on AA (arachidonic acid)-activated Ca2+ channels. We studied the effects of AA on membrane conductance of rat liver cells using whole-cell patch clamping. We found no evidence that concentrations of AA in the physiological range could activate Ca2+-permeable channels in either H4IIE liver cells or rat hepatocytes. However, AA (1–10 μM) did inhibit (IC50=2.4±0.1 μM) Ca2+ inflow through SOCs (ISOC) initiated by intracellular application of Ins(1,4,5)P3 in H4IIE cells. Pre-incubation with AA did not inhibit ISOC development, but decreased maximal amplitude of the current. Iso-tetrandrine, widely used to inhibit receptor-activation of phospholipase A2, and therefore AA release, inhibited ISOC directly in H4IIE cells. It is concluded that (i) in rat liver cells, AA does not activate an AA-regulated Ca2+-permeable channel, but does inhibit SOCs, and (ii) iso-tetrandrine and tetrandrine are effective blockers of CRAC (Ca2+-release-activated Ca2+) channel-like SOCs. These results indicate that AA-activated Ca2+-permeable channels do not contribute to hormone-induced increases or oscillations in [Ca2+]cyt in liver cells. However, AA may be a physiological modulator of Ca2+ inflow in these cells.

Evidence that 2-aminoethyl diphenylborate is a novel inhibitor of store-operated Ca2+ channels in liver cells, and acts through a mechanism which does not involve inositol trisphosphate receptors

2001 ◽  
Vol 354 (2) ◽  
pp. 285-290 ◽  
Author(s):  
Roland B. GREGORY ◽  
Grigori RYCHKOV ◽  
Greg J. BARRITT
Keyword(s):  
Hepatoma Cell ◽  
Regulatory Protein ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Detectable Effect ◽  
Hepatoma Cell Line ◽  
Isolated Rat Hepatocytes ◽  
Fura 2 ◽  
Ca2 Channels

The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P3 receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P3 receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647–1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10µM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) had been established, the kinetics of the decrease in [Ca2+]cyt were identical with those induced by the addition of 50µM Gd3+ (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P3 to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P3-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P3 receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P3 receptors in the activation of SOCs in other cell types is briefly discussed.

scholarly journals Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

2016 ◽  
Author(s):  
Christopher D Richardson ◽  
Grahm J Ray ◽  
Jacob E Corn
Keyword(s):  
Cell Lines ◽  
Gene Disruption ◽  
Genomic Sequence ◽  
Cell Types ◽  
Polyploid Cell ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Specific Manner ◽  
A Cell ◽  
Different Cell Types

Cas9 endonuclease can be targeted to genomic sequences by varying the sequence of the single guide RNA (sgRNA). The activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines, or using inefficient sgRNAs, can require extensive downstream screening to identify homozygous clones. We have found that linear, non-homologous oligonucleotide DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation greatly increases the frequency of clones with homozygous gene disruptions, even in polyploid cell lines, and rescues otherwise ineffective sgRNAs. The mechanism of enhanced gene disruption differs between human cell lines, stimulating deletion of genomic sequence and/or insertion of non-homologous oligonucleotide DNA at the edited locus in a cell line specific manner. Thus, the addition of non-homologous DNA appears to drive cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

scholarly journals Cell type- and stage-specific expression of Otx2 is coordinated by a cohort of transcription factors and multiple cis-regulatory modules in the retina

2019 ◽  
Author(s):  
Candace Chan ◽  
Nicolas Lonfat ◽  
Rong Zhao ◽  
Alexander Davis ◽  
Liang Li ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Cell Types ◽  
Retinal Cell ◽  
Cell Type ◽  
Specific Expression ◽  
Regulatory Modules ◽  
Specific Manner ◽  
A Cell

AbstractTranscription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is an advantageous strategy. However, the mechanisms that control the activation of TFs in different cell types and at different stages of development remain unclear. The neural retina serves as a model of the development of a complex tissue. We used this system to analyze how expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during retinogenesis. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. These include mature bipolar interneurons, photoreceptors, and retinal progenitor/precursor cells. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages, as revealed by ATAC-seq and DNase-seq analyses. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2 in a cell type- and stage-specific manner in the retina.

scholarly journals OCI-5/GPC3, a Glypican Encoded by a Gene That Is Mutated in the Simpson-Golabi-Behmel Overgrowth Syndrome, Induces Apoptosis in a Cell Line–specific Manner

1998 ◽  
Vol 141 (6) ◽  
pp. 1407-1414 ◽  
Author(s):  
Alfonso Dueñas Gonzalez ◽  
Mitsunori Kaya ◽  
Wen Shi ◽  
Howard Song ◽  
Joseph R. Testa ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Types ◽  
Glycosyl Phosphatidylinositol ◽  
Overgrowth Syndrome ◽  
3T3 Fibroblasts ◽  
Specific Manner ◽  
A Cell ◽  
Nih 3T3 ◽  
Insight Into ◽  
Mcf 7

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3–induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line– specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.

scholarly journals Alternative Splicing of the Fibronectin EIIIB Exon Depends on Specific TGCATG Repeats

1998 ◽  
Vol 18 (7) ◽  
pp. 3900-3906 ◽  
Author(s):  
Lee P. Lim ◽  
Phillip A. Sharp
Keyword(s):  
Point Mutations ◽  
Cell Types ◽  
Sr Protein ◽  
Multicomponent Complex ◽  
Evolutionarily Conserved ◽  
Specific Manner ◽  
Alternatively Spliced ◽  
A Cell ◽  
Cell Type Specific ◽  
Quantitative Changes

ABSTRACT The fibronectin EIIIB exon is alternatively spliced in a cell-type-specific manner, and TGCATG repeats in the intron downstream of EIIIB have been implicated in this regulation. Analysis of the intron sequence from several vertebrates shows that the pattern of repeats in the 3′ half of the intron is evolutionarily conserved. Point mutations in certain highly conserved repeats greatly reduce EIIIB inclusion, suggesting that a multicomponent complex may recognize the repeats. Expression of the SR protein SRp40, SRp20, or ASF/SF2 stimulates EIIIB inclusion. Studies of the interplay between mutations in the repeats and SRp40-stimulated inclusion suggest that the repeats are recognized in many, if not all, cell types, and that EIIIB inclusion may be regulated by quantitative changes in multiple factors.

Abstract 136: Distinct Temporal Requirements for Scl to Repress Cardiomyogenesis in Endothelial-derived Cells in Hemogenic Tissues and the Heart

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Ben Van Handel ◽  
Tonis Org ◽  
Amelie Montel-Hagen ◽  
Haruko Nakano ◽  
Atsushi Nakano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Window ◽  
Ectopic Expression ◽  
Cell Types ◽  
Yolk Sac ◽  
Lineage Tracing ◽  
Cell Type ◽  
Specific Manner ◽  
A Cell ◽  
Cell Type Specific

Identification of precursors with the capacity to generate cardiomyocytes is critical for advancing cardiac regenerative medicine. By analyzing knockout embryos for the bHLH factor Scl, we demonstrated that endothelial cells in hematopoietic tissues and the heart possess latent cardiomyogenic capacity. Furthermore, analysis of tamoxifen-inducible Rosa26-Cre ERT2 Scl fl/fl embryos suggested that the time window during which Scl is required for cardiac repression extends later in the heart versus the yolk sac. However, the cell types in which Scl acts remained elusive. We then deleted Scl in a cell-type specific manner in early mesoderm using Mesp1-Cre and in endothelial cells using Tie2-Cre. Lineage tracing in Mesp1-Cre Rosa26-YFP embryos demonstrated that at E9.5, a large majority of hematopoietic and endothelial cells in the yolk sac and heart were labeled. Moreover, deletion of Scl in Mesp1-Cre Scl fl/fl embryos phenocopied the germline knockout, essentially abrogating hematopoiesis and promoting the emergence of CD31 + PDGFRα + cardiomyogenic precursors and ectopic expression of the cardiomyocyte genes Myl7 and Tnnt2 in yolk sac vasculature. In contrast, deletion of Scl after endothelium had been specified in Tie2-Cre Scl fl/fl embryos did not grossly affect yolk sac hematopoiesis, nor did it induce ectopic cardiomyogenesis in hemogenic tissues. However, endothelial-derived cells in the hearts of Tie2-Cre Scl fl/fl embryos evidenced profound expansion of CD31 + PDGFRα + cardiogenic precursors at E11.5 and E13.5, as well as displayed dramatic upregulation of Myl7 and Tnnt2 , showing that the requirement for Scl to repress the cardiomyogenic program extends longer in endothelial derivatives in the heart than in the yolk sac. These data demonstrate that endocardial-derived cells in the heart retain latent cardiomyogenic potential until mid-gestation and nominate Scl as a critical regulator of endocardial fate.

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