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Sample records for ryanodine receptor isoform

  1. Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

    PubMed

    Lai, F A; Liu, Q Y; Xu, L; el-Hashem, A; Kramarcy, N R; Sealock, R; Meissner, G

    1992-08-01

    The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR. PMID:1325114

  2. The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2+

    PubMed Central

    Rybalchenko, Volodymyr; Grillo, Michael A.; Gastinger, Matthew J.; Rybalchenko, Nataliya; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Ca2+ release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca2+-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical “non-genomic” effects mediated by estrogen receptors (ER) include rapid Ca2+ release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of co-localization between RyR type 2 (RyR2) and ER type β (ERβ) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ERβ (ERβ1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca2+] concentrations of 100 nM, suggesting a synergistic action of ERβ1 and Ca2+ in RyR activation, and a potential contribution to Ca2+-induced Ca2+ release rather than to basal intracellular Ca2+ concentration level at rest. This RyR/ERβ interaction has potential effects on cellular physiology, including roles of shorter ERβ isoforms and modulation of the RyR/ERβ complexes by exogenous estrogens. PMID:19899956

  3. Identification of ryanodine receptor isoforms in prostate DU-145, LNCaP, and PWR-1E cells.

    PubMed

    Kobylewski, Sarah E; Henderson, Kimberly A; Eckhert, Curtis D

    2012-08-24

    The ryanodine receptor (RyR) is a large, intracellular calcium (Ca(2+)) channel that is associated with several accessory proteins and is an important component of a cell's ability to respond to changes in the environment. Three isoforms of the RyR exist and are well documented for skeletal and cardiac muscle and the brain, but the isoforms in non-excitable cells are poorly understood. The aggressiveness of breast cancers in women has been positively correlated with the expression of the RyR in breast tumor tissue, but it is unknown if this is limited to specific isoforms. Identification and characterization of RyRs in cancer models is important in understanding the role of the RyR channel complex in cancer and as a potential therapeutic target. The objective of this report was to identify the RyR isoforms expressed in widely used prostate cancer cell lines, DU-145 and LNCaP, and the non-tumorigenic prostate cell line, PWR-1E. Oligonucleotide primers specific for each isoform were used in semi-quantitative and real-time PCR to determine the identification and expression levels of the RyR isoforms. RyR1 was expressed in the highest amount in DU-145 tumor cells, expression was 0.48-fold in the non-tumor cell line PWR-1E compared to DU-145 cells, and no expression was observed in LNCaP tumor cells. DU-145 cells had the lowest expression of RyR2. The expression was 26- and 15-fold higher in LNCaP and PWR-1E cells, respectively. RyR3 expression was not observed in any of the cell lines. All cell types released Ca(2+) in response to caffeine showing they had functional RyRs. Total cellular RyR-associated Ca(2+) release is determined by both the number of activated RyRs and its accessory proteins which modulate the receptor. Our results suggest that the correlation between the expression of the RyR and tumor aggression is not related to specific RyR isoforms, but may be related to the activity and number of receptors. PMID:22846571

  4. Expression and function of ryanodine receptors in nonexcitable cells.

    PubMed

    Bennett, D L; Cheek, T R; Berridge, M J; De Smedt, H; Parys, J B; Missiaen, L; Bootman, M D

    1996-03-15

    We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses. PMID:8626432

  5. Physiological differences between the alpha and beta ryanodine receptors of fish skeletal muscle.

    PubMed Central

    O'Brien, J; Valdivia, H H; Block, B A

    1995-01-01

    Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (alpha and beta) by comparing SR preparations from specialized fish muscles that express the alpha isoform alone to preparations from muscles containing both alpha and beta. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish alpha isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the alpha RYR were achieved from 1 to 10 microM free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The beta isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the beta and cardiac isoforms from the alpha isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the beta and cardiac RYRs are not identical, however. [3H]ryanodine binding by the alpha RYR was selectively inhibited by 100 microM tetracaine, whereas cardiac and beta RYRs were much less affected. Tetracaine can thus be used to separate the properties of the alpha and beta RYRs in preparations in which both are present. The distinct physiological properties of the alpha and beta RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca(2+)-coupled channels within a single triad junction. PMID:7696500

  6. Different Involvement of Type 1, 2, and 3 Ryanodine Receptors in Memory Processes

    ERIC Educational Resources Information Center

    Galeotti, Nicoletta; Quattrone, Alessandro; Vivoli, Elisa; Norcini, Monica; Bartolini, Alessandro; Ghelardini, Carla

    2008-01-01

    The administration of the ryanodine receptor (RyR) agonist 4-Cmc (0.003-9 nmol per mouse intracerebroventricularly [i.c.v.]) ameliorated memory functions, whereas the RyR antagonist ryanodine (0.0001-1 nmol per mouse i.c.v.) induced amnesia in the mouse passive avoidance test. The role of the type 1, 2, and 3 RyR isoforms in memory processes was…

  7. Arrhythmogenic mechanisms in ryanodine receptor channelopathies.

    PubMed

    Zhao, Yan-Ting; Valdivia, Carmen R; Gurrola, Georgina B; Hernández, Jonathan J; Valdivia, Héctor H

    2015-01-01

    Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of calcium ions (Ca(2+)) necessary to induce contraction of cardiac and skeletal muscle cells. In their intracellular environment, RyR channels are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca(2+)) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias) or cellular remodeling. The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in RYR2, the gene encoding for the cardiac isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by RyR2 channels harboring mutations linked to CPVT. Most CPVT mutations characterized to date endow RyR2 channels with a gain of function, resulting in hyperactive channels that release Ca(2+) spontaneously, especially during diastole. The spontaneous Ca(2+) release is extruded by the electrogenic Na(+)/Ca(2+) exchanger, which depolarizes the external membrane (delayed afterdepolarization or DAD) and may trigger untimely action potentials. However, a rare set of CPVT mutations yield RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli, and it is unclear whether these channels release Ca(2+) spontaneously during diastole. We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to RyR2 loss-of-function mutations. PMID:25480325

  8. Catecholamime Interactions with the Cardiac Ryanodine Receptor

    NASA Astrophysics Data System (ADS)

    Klipp, Robert Carl

    The cardiac ryanodine receptor (RyR2) is a Ca2+ ion channel found in the sarcoplasmic reticulum (SR), an intracellular membranous Ca2+ storage system. It is well known that a destabilization of RyR2 can lead to a Ca2+ flux out of the SR, which results in an overload of intracellular Ca2+; this can also lead to arrhythmias and heart failure. The catecholamines play a large role in the regulation of RyR2; stimulation of the beta-adrenergic receptor on the cell membrane can lead to a hyperphosphorylation of RyR2, making it more leaky to Ca2+. We have previously shown that strong electron donors will inhibit RyR2. It is hypothesized that the catecholamines, sharing a similar structure with other proven inhibitors of RyR2, will also inhibit RyR2. Here we confirm this hypothesis and show for the first time that the catecholamines, isoproterenol and epinephrine, act as strong electron donors and inhibit RyR2 activity at the single channel level. This data suggests that the catecholamines can influence RyR2 activity at two levels. This offers promising insight into the potential development of a new class of drugs to treat heart failure and arrhythmia; ones that can both prevent the hyperphosphorylation of RyR2 by blocking the beta-adrenergic receptor, but can also directly inhibit the release of Ca2+ from RyR2.

  9. Structure of a mammalian ryanodine receptor

    PubMed Central

    Zalk, Ran; Clarke, Oliver B.; des Georges, Amédée; Grassucci, Robert A.; Reiken, Steven; Mancia, Filippo; Hendrickson, Wayne A.; Frank, Joachim; Marks, Andrew R.

    2014-01-01

    Ryanodine receptors (RyRs) mediate rapid release of calcium (Ca2+) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here, we report the closed-state structure of the 2.3 MDa complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle cryo-electron microscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3939 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane (6TM) ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca2+. PMID:25470061

  10. Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1).

    PubMed Central

    Du, G G; Oyamada, H; Khanna, V K; MacLennan, D H

    2001-01-01

    Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1. PMID:11695996

  11. Caveolin-3 is adjacent to a group of extradyadic ryanodine receptors.

    PubMed

    Scriven, David R L; Klimek, Agnieszka; Asghari, Parisa; Bellve, Karl; Moore, Edwin D W

    2005-09-01

    Caveolae are present in almost all cells and concentrate a wide variety of signaling molecules, receptors, transporters, and ion pumps. We have investigated the distribution of the ryanodine receptor, the Na(+)/Ca(2+) exchanger, the predominant Na(+) channel isoform rH1, and the L-type calcium channel, Ca(v)1.2, relative to the muscle-specific caveolin isoform, caveolin-3, in adult rat ventricular myocytes. Three-dimensional immunofluorescence images were deconvolved and analyzed. Caveolin-3 colocalizes with all of these molecules at the surface of the cell, but there is no significant colocalization between caveolin-3 and either the Na(+)/Ca(2+) exchanger or the Na(+) channel in the cell interior. The distribution of the surface colocalization indicates that the caveolae that colocalize with each molecule form distinct populations. This organization indicates that there are multiple populations of caveolae separable by location and occupants. In the interior of the cell, caveolin-3 shows a marked colocalization with a population of ryanodine receptors that are separate from those within the dyad. Because of their location, the signaling molecules contained within these caveolae may have preferred access to the neighboring nondyadic ryanodine receptors. PMID:15980179

  12. CCDI: a new ligand that modulates mammalian type 1 ryanodine receptor (RyR1)

    PubMed Central

    Tian, Chengju; Shao, Chun Hong; Padanilam, Christina; Ezell, Edward; Singh, Jaipaul; Kutty, Shelby; Bidasee, Keshore R

    2014-01-01

    Background and Purpose Ryanodine receptors (RyRs) are Ca2+-release channels on the sarco(endo)plasmic reticulum that modulate a wide array of physiological functions. Three RyR isoforms are present in cells: RyR1, RyR2 and RyR3. To date, there are no reports on ligands that modulate RyR in an isoform-selective manner. Such ligands are not only valuable research tools, but could serve as intermediates for development of therapeutics. Experimental approach Pyrrole-2-carboxylic acid and 1,3-dicyclohexylcarbodiimide were allowed to react in carbon tetrachloride for 24 h at low temperatures and pressures. The chemical structures of the two products isolated were elucidated using NMR spectrometry, mass spectrometry and elemental analyses. [3H]-ryanodine binding, lipid bilayer and time-lapsed confocal imaging were used to determine their effects on RyR isoforms. Key results The major product, 2-cyclohexyl-3-cyclohexylimino-2, 3, dihydro–pyrrolo[1,2-c]imidazol-1-one (CCDI) dose-dependently potentiated Ca2+-dependent binding of [3H]-ryanodine to RyR1, with no significant effects on [3H]-ryanodine binding to RyR2 or RyR3. CCDI also reversibly increased the open probability (Po) of RyR1 with minimal effects on RyR2 and RyR3. CCDI induced Ca2+ transients in C2C12 skeletal myotubes, but not in rat ventricular myocytes. This effect was blocked by pretreating cells with ryanodine. The minor product 2-cyclohexyl-pyrrolo[1,2-c]imidazole-1,3-dione had no effect on either [3H]-ryanodine binding or Po of RyR1, RyR2 and RyR3. Conclusions and implications A new ligand that preferentially modulates RyR1 was identified. In addition to being an important research tool, the pharmacophore of this small molecule could serve as a template for the synthesis of other isoform-selective modulators of RyRs. PMID:24819467

  13. Functional ryanodine receptors in the membranes of neurohypophysial secretory granules

    PubMed Central

    McNally, James M.; Custer, Edward E.; Ortiz-Miranda, Sonia; Woodbury, Dixon J.; Kraner, Susan D.; Salzberg, Brian M.

    2014-01-01

    Highly localized Ca2+ release events have been characterized in several neuronal preparations. In mouse neurohypophysial terminals (NHTs), such events, called Ca2+ syntillas, appear to emanate from a ryanodine-sensitive intracellular Ca2+ pool. Traditional sources of intracellular Ca2+ appear to be lacking in NHTs. Thus, we have tested the hypothesis that large dense core vesicles (LDCVs), which contain a substantial amount of calcium, represent the source of these syntillas. Here, using fluorescence immunolabeling and immunogold-labeled electron micrographs of NHTs, we show that type 2 ryanodine receptors (RyRs) are localized specifically to LDCVs. Furthermore, a large conductance nonspecific cation channel, which was identified previously in the vesicle membrane and has biophysical properties similar to that of an RyR, is pharmacologically affected in a manner characteristic of an RyR: it is activated in the presence of the RyR agonist ryanodine (at low concentrations) and blocked by the RyR antagonist ruthenium red. Additionally, neuropeptide release experiments show that these same RyR agonists and antagonists modulate Ca2+-elicited neuropeptide release from permeabilized NHTs. Furthermore, amperometric recording of spontaneous release events from artificial transmitter-loaded terminals corroborated these ryanodine effects. Collectively, our findings suggest that RyR-dependent syntillas could represent mobilization of Ca2+ from vesicular stores. Such localized vesicular Ca2+ release events at the precise location of exocytosis could provide a Ca2+ amplification mechanism capable of modulating neuropeptide release physiologically. PMID:24863930

  14. Spatial localization of ryanodine receptors in human cardiac cells.

    PubMed

    Vallmitjana, A; Nolla, C; Herraiz-Martínez, A; Hove-Madsen, L; Benítez, R

    2015-08-01

    We present a novel image processing method to determine the location of ryanodine receptors in cardiac cells. A semi-automatic manual validation by an expert has been used in order to establish the performance of the segmentation method. The approach provides high accuracy under different experimental conditions and it is robust to common sources of noise including experimental, molecular and biological fluctuations. PMID:26737732

  15. Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation.

    PubMed

    Adasme, Tatiana; Haeger, Paola; Paula-Lima, Andrea C; Espinoza, Italo; Casas-Alarcón, M Mercedes; Carrasco, M Angélica; Hidalgo, Cecilia

    2011-02-15

    Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation. PMID:21282625

  16. Involvement of ryanodine receptors in neurotrophin-induced hippocampal synaptic plasticity and spatial memory formation

    PubMed Central

    Adasme, Tatiana; Haeger, Paola; Paula-Lima, Andrea C.; Espinoza, Italo; Casas-Alarcón, M. Mercedes; Carrasco, M. Angélica; Hidalgo, Cecilia

    2011-01-01

    Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation. PMID:21282625

  17. Inhibitory ryanodine prevents ryanodine receptor-mediated Ca²⁺ release without affecting endoplasmic reticulum Ca²⁺ content in primary hippocampal neurons.

    PubMed

    Adasme, Tatiana; Paula-Lima, Andrea; Hidalgo, Cecilia

    2015-02-27

    Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca(2+)-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca(2+) release and endoplasmic reticulum Ca(2+) depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca(2+) release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca(2+) release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca(2+) levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca(2+) release, preserves ER Ca(2+) levels and thus constitutes a sound strategy to suppress neuronal RyR function. PMID:25623539

  18. Ryanodine receptors are uncoupled from contraction in rat vena cava.

    PubMed

    Tykocki, N R; Thompson, J M; Jackson, W F; Watts, S W

    2013-02-01

    Ryanodine receptors (RyR) are Ca(2+)-sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors of SR Ca(2+) release and smooth muscle excitation-contraction coupling. While the relationship between RyR activation and contraction is well characterized in arteries, little is known about the role of RyR in excitation-contraction coupling in veins. We hypothesized that RyR are present and directly coupled to contraction in rat aorta (RA) and vena cava (RVC). RA and RVC expressed mRNA for all 3 RyR subtypes, and immunofluorescence showed RyR protein was present in RA and RVC smooth muscle cells. RA and RVC rings contracted when Ca(2+) was re-introduced after stores depletion with thapsigargin (1μM), indicating both tissues contained intracellular Ca(2+) stores. To assess RyR function, contraction was then measured in RA and RVC exposed to the RyR activator caffeine (20mM). In RA, caffeine caused contraction that was attenuated by the RyR antagonists ryanodine (10μM) and tetracaine (100μM). However, caffeine (20mM) did not contract RVC. We next measured contraction and intracellular Ca(2+) (Ca(2+)(i)) simultaneously in RA and RVC exposed to caffeine. While caffeine increased Ca(2+)(i) and contracted RA, it had no significant effect on Ca(2+)(i) or contraction in RVC. These data suggest that ryanodine receptors, while present in both RA and RVC, are inactive and uncoupled from Ca(2+) release and contraction in RVC. PMID:23177664

  19. A procedure for purification of the ryanodine receptor from skeletal muscle

    SciTech Connect

    Hawkes, M.J.; Diaz-Munoz, M.; Hamilton, S.L. )

    1989-01-01

    In this paper, we describe a simple and reproducible method for purifying large quantities of ryanodine receptor from skeletal muscle membranes. The procedure involves the use of ion exchange chromatography and sucrose gradient centrifugation to purify the protein which has been identified as the calcium release protein of the sarcoplasmic reticulum. Addition of micromolar quantities of unlabeled ryanodine prior to solubilization and throughout the isolation procedure appears to stabilize the tetrameric structure of the ryanodine receptor. The purified receptor, consisting predominantly of a 400K polypeptide on SDS-PAGE, binds ({sup 3}H)ryanodine with a binding affinity similar to that in membranes. Overall recovery of ryanodine binding activity was 21% of the initial activity with a 30-fold purification of the receptor.

  20. The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis.

    PubMed

    Tunwell, R E; Wickenden, C; Bertrand, B M; Shevchenko, V I; Walsh, M B; Allen, P D; Lai, F A

    1996-09-01

    Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree. PMID:8809036

  1. Ryanodine receptors contribute to the induction of ischemic tolerance.

    PubMed

    Nakamura-Maruyama, Emi; Miyamoto, Osamu; Okabe, Naohiko; Himi, Naoyuki; Feng, Lu; Narita, Kazuhiko; Keep, Richard F; Yamamoto, Tohru; Nakamura, Takehiro

    2016-04-01

    Ischemic tolerance (IT) is induced by a variety of insults to the brain (e.g., nonfatal ischemia, heat and hypoxia) and it provides a strong neuroprotective effect. Although the mechanisms are still not fully elucidated, Ca(2+) is regarded as a key mediator of IT. Ryanodine receptors (RyRs) are located in the sarcoplasmic/endoplasmic reticulum membrane and are responsible for the release of Ca(2+) from intracellular stores. In brain, neuronal RyRs are thought to play a role in various neuropathological conditions, including ischemia. The purpose of the present study was to investigate the involvement of RyRs in IT. Pretreatment with a RyR antagonist, dantrolene (25mg/kg, i.p), blocked IT in a gerbil global ischemia model, while a RyR agonist, caffeine (100mg/kg, i.p), stimulated the production of IT. In vitro, using rat hippocampal cells, short-term oxygen/glucose deprivation induced preconditioning and RyR antagonists, dantrolene (50 and 100μM) and ryanodine (100 and 200μM) prevented it. RyR protein and mRNA levels were transiently decreased after induction of IT. These results suggest that RyRs are involved in the induction of ischemic tolerance. PMID:26930163

  2. Identification of a critical region in the Drosophila ryanodine receptor that confers sensitivity to diamide insecticides.

    PubMed

    Tao, Yong; Gutteridge, Steven; Benner, Eric A; Wu, Lihong; Rhoades, Daniel F; Sacher, Matthew D; Rivera, Michel A; Desaeger, Johan; Cordova, Daniel

    2013-09-01

    Anthranilic diamides, which include the new commercial insecticide, chlorantraniliprole, are an exciting new class of chemistry that target insect ryanodine receptors. These receptors regulate release of stored intracellular calcium and play a critical role in muscle contraction. As with insects, nematodes express ryanodine receptors and are sensitive to the plant alkaloid, ryanodine. However the plant parasitic nematode, Meloidogyne incognita, is insensitive to anthranilic diamides. Expression of a full-length Drosophila melanogaster ryanodine receptor in an insect cell line confers sensitivity to the receptor agents, caffeine and ryanodine along with nanomolar sensitivity to anthranilic diamides. Replacement of a 46 amino acid segment in a highly divergent region of the Drosophila C-terminus with that from Meloidogyne results in a functional RyR which lack sensitivity to diamide insecticides. These findings indicate that this region is critical to diamide sensitivity in insect ryanodine receptors. Furthermore, this region may contribute to our understanding of the differential selectivity diamides exhibit for insect over mammalian ryanodine receptors. PMID:23806522

  3. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca2+ release channel complex.

    PubMed

    Anderson, K; Lai, F A; Liu, Q Y; Rousseau, E; Erickson, H P; Meissner, G

    1989-01-15

    Using density gradient centrifugation and [3H]ryanodine as a specific marker, the ryanodine receptor-Ca2+ release channel complex from Chaps-solubilized canine cardiac sarcoplasmic reticulum (SR) has been purified in the form of an approximately 30 S complex, comprised of Mr approximately 400,000 polypeptides. Purification resulted in a specific activity of approximately 450 pmol bound ryanodine/mg of protein, a 60-70% recovery of ryanodine binding activity, and retention of the high affinity ryanodine binding site (KD = 3 nM). Negative stain electron microscopy revealed a 4-fold symmetric, four-leaf clover structure, which could fill a box approximately 30 x 30 nm and was thus morphologically similar to the SR-transverse-tubule, junctionally associated foot structure. The structural, sedimentation, and ryanodine binding data strongly suggest there is one high affinity ryanodine binding site/30 S complex, comprised of four Mr approximately 400,000 subunits. Upon reconstitution into planar lipid bilayers, the purified complex exhibited a Ca2+ conductance (70 pS in 50 mM Ca2+) similar to that of the native cardiac Ca2+ release channel (75 pS). The reconstituted complex was also found to conduct Na+ (550 pS in 500 mM Na+) and often to display complex Na+ subconducting states. The purified channel could be activated by micromolar Ca2+ or millimolar ATP, inhibited by millimolar Mg2+ or micromolar ruthenium red, and modified to a long-lived open subconducting state by ryanodine. The sedimentation, subunit composition, morphological, and ryanodine binding characteristics of the purified cardiac ryanodine receptor-Ca2+ release channel complex were similar to those previously described for the purified ryanodine receptor-Ca2+ release channel complex from fast-twitch skeletal muscle. PMID:2463249

  4. Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle.

    PubMed Central

    Marty, I; Robert, M; Villaz, M; De Jongh, K; Lai, Y; Catterall, W A; Ronjat, M

    1994-01-01

    Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Analysis of the sedimentation behavior of the solubilized proteins showed the existence of a population of alpha 1 subunits of the dihydropyridine receptor which cosedimented with the ryanodine receptor. Solubilized proteins were immunoprecipitated with antibodies directed against either the ryanodine receptor or the alpha 1, alpha 2, or beta subunits of the dihydropyridine receptor. Immunoprecipitated proteins were identified by Western blot analysis and by specific labeling with [3H]ryanodine or [3H]PN200-110. Immunoprecipitation of the solubilized proteins with antibodies directed against the dihydropyridine receptor led to the coimmunoprecipitation of the ryanodine receptor. Conversely, immunoprecipitation with antibodies directed against the ryanodine receptor led to an immune complex containing both receptors, but these antibodies were unable to precipitate purified dihydropyridine receptor. These results demonstrate that ryanodine receptor and dihydropyridine receptor are present in the triad membrane preparation in a complex which may play an important role in excitation-contraction coupling. Images PMID:8134386

  5. Coupled gating between cardiac calcium release channels (ryanodine receptors).

    PubMed

    Marx, S O; Gaburjakova, J; Gaburjakova, M; Henrikson, C; Ondrias, K; Marks, A R

    2001-06-01

    Excitation-contraction coupling in heart muscle requires the activation of Ca(2+)-release channels/type 2 ryanodine receptors (RyR2s) by Ca(2+) influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca(2+) release. PMID:11397781

  6. Selective expression of the type 3 isoform of ryanodine receptor Ca{sup 2+} release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits

    SciTech Connect

    Conti, Antonio; Reggiani, Carlo; Sorrentino, Vincenzo . E-mail: v.sorrentino@unisi.it

    2005-11-11

    The expression pattern of the RyR3 isoform of Ca{sup 2+} release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.

  7. Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

    PubMed

    Matecki, Stefan; Dridi, Haikel; Jung, Boris; Saint, Nathalie; Reiken, Steven R; Scheuermann, Valérie; Mrozek, Ségolène; Santulli, Gaetano; Umanskaya, Alisa; Petrof, Basil J; Jaber, Samir; Marks, Andrew R; Lacampagne, Alain

    2016-08-01

    Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with β-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention. PMID:27457930

  8. Architecture and conformational switch mechanism of the ryanodine receptor.

    PubMed

    Efremov, Rouslan G; Leitner, Alexander; Aebersold, Ruedi; Raunser, Stefan

    2015-01-01

    Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel. PMID:25470059

  9. Fluorescent probes for insect ryanodine receptors: candidate anthranilic diamides.

    PubMed

    Wang, Yi; Guo, Lei; Qi, Suzhen; Zhang, Hao; Liu, Kechang; Liu, Ruiquan; Liang, Pei; Casida, John E; Liu, Shangzhong

    2014-01-01

    Diamide insecticides with high efficacy against pests and good environmental safety are broadly applied in crop protection. They act at a poorly-defined site in the very complex ryanodine (Ry) receptor (RyR) potentially accessible to a fluorescent probe. Two N-propynyl analogs of the major anthranilic diamide insecticides chlorantraniliprole (Chlo) and cyantraniliprole (Cyan) were accordingly synthesized and converted into two fluorescent ligands by click reaction coupling with 3-azido-7-hydroxy-2H-chromen-2-one. The new diamide analogs and fluorescent ligands were shown to be nearly as potent as Chlo and Cyan in inhibition of [3H]Chlo binding and stimulation of [3H]Ry binding in house fly thoracic muscle RyR. Although the newly synthesized compounds had only moderate activity in insect larvicidal activity assays, their high in vitro potency in a validated insect RyR binding assay encourages further development of fluorescent probes for insect RyRs. PMID:24699151

  10. Drosophila Ryanodine Receptors Mediate General Anesthesia by Halothane

    PubMed Central

    Gao, Shuying; Sandstrom, David J.; Smith, Harold E.; High, Brigit; Marsh, Jon W.; Nash, Howard A.

    2012-01-01

    Background Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and we hypothesized that it has a central role in anesthesia. Methods Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca2+ imaging and patch clamp electrophysiology. Results Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca2+ efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca2+ imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca2+ flux that is altered in dRyr mutants and correlates with strong hyperpolarization. Conclusions In Drosophila, neurally-expressed dRyr mediates a substantial proportion of halothane's anesthetic effects in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. Our results provide support for Ryr as an important mediator of immobilization by volatile anesthetics. PMID:23254148

  11. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

    PubMed

    Woodier, Jason; Rainbow, Richard D; Stewart, Alan J; Pitt, Samantha J

    2015-07-10

    Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  12. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

    PubMed Central

    Woodier, Jason; Rainbow, Richard D.; Stewart, Alan J.; Pitt, Samantha J.

    2015-01-01

    Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  13. Ryanodine receptor physiology and its role in disease.

    PubMed

    Lanner, Johanna T

    2012-01-01

    The ryanodine receptors (RyRs) is the major intracellular Ca(2+) release channel localized in the plasma membrane of the endoplasmatic/sarcoplasmatic reticulum. RyR-mediated Ca(2+) release is crucial for every heart beat and skeletal muscle contraction and also important in learning and memory. Given the important role RyR has in physiological functions it is not surprising that dysregulation and impaired RyR channel function contributes to severe pathologies e.g. cardiac arrhythmias and Alzheimer's disease. Mutations in the RyR channels are associated with a number of human disorders e.g. malignant hyperthermia (MH) and central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia (ARVD). RyRs are modulated directly and indirectly by various ions, small molecules and proteins and RyR structure and function are expected to be defined within this macromolecular set of interactions. This article discusses the physiological function of RyR and examines its role in disorders and diseases. PMID:22453944

  14. Insect Ryanodine Receptor: Distinct But Coupled Insecticide Binding Sites for [N-C3H3]Chlorantraniliprole, Flubendiamide, and [3H]Ryanodine

    PubMed Central

    Isaacs, André K.; Qi, Suzhen; Sarpong, Richmond; Casida, John E.

    2015-01-01

    Radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole was synthesized at high specific activity and compared with phthalic diamide insecticide flubendiamide and [3H]ryanodine in radioligand binding studies with house fly muscle membranes to provide the first direct evidence with a native insect ryanodine receptor that the major anthranilic and phthalic diamide insecticides bind at different allosterically coupled sites, i.e. there are three distinct Ca2+-release channel targets for insecticide action. PMID:22856329

  15. Marked Sexual Dimorphism in the Role of the Ryanodine Receptor in a Model of Pain Chronification in the Rat.

    PubMed

    Ferrari, Luiz F; Khomula, Eugen V; Araldi, Dionéia; Levine, Jon D

    2016-01-01

    Hyperalgesic priming, an estrogen dependent model of the transition to chronic pain, produced by agonists at receptors that activate protein kinase C epsilon (PKCε), occurs in male but not in female rats. However, activation of second messengers downstream of PKCε, such as the ryanodine receptor, induces priming in both sexes. Since estrogen regulates intracellular calcium, we investigated the interaction between estrogen and ryanodine in the susceptibility to develop priming in females. The lowest dose of ryanodine able to induce priming in females (1 pg) is 1/100,000(th) that needed in males (100 ng), an effect dependent on the activation of ryanodine receptors. Treatment of female rats with antisense to estrogen receptor alpha (ERα), but not beta (ERβ), mRNA, prevented the induction of priming by low dose ryanodine, and the ERα agonist, PPT, induced ryanodine receptor-dependent priming. In vitro application of ryanodine in low concentration (2 nM) to small DRG neurons cultured from females, significantly potentiated calcium release via ryanodine receptors induced by caffeine. This effect was only observed in IB4+ neurons, cultured in the presence of β-estradiol or PPT. Our results demonstrate a profound regulatory role of ERα in ryanodine receptor-dependent transition to chronic pain. PMID:27499186

  16. Marked Sexual Dimorphism in the Role of the Ryanodine Receptor in a Model of Pain Chronification in the Rat

    PubMed Central

    Ferrari, Luiz F.; Khomula, Eugen V.; Araldi, Dionéia; Levine, Jon D.

    2016-01-01

    Hyperalgesic priming, an estrogen dependent model of the transition to chronic pain, produced by agonists at receptors that activate protein kinase C epsilon (PKCε), occurs in male but not in female rats. However, activation of second messengers downstream of PKCε, such as the ryanodine receptor, induces priming in both sexes. Since estrogen regulates intracellular calcium, we investigated the interaction between estrogen and ryanodine in the susceptibility to develop priming in females. The lowest dose of ryanodine able to induce priming in females (1 pg) is 1/100,000th that needed in males (100 ng), an effect dependent on the activation of ryanodine receptors. Treatment of female rats with antisense to estrogen receptor alpha (ERα), but not beta (ERβ), mRNA, prevented the induction of priming by low dose ryanodine, and the ERα agonist, PPT, induced ryanodine receptor-dependent priming. In vitro application of ryanodine in low concentration (2 nM) to small DRG neurons cultured from females, significantly potentiated calcium release via ryanodine receptors induced by caffeine. This effect was only observed in IB4+ neurons, cultured in the presence of β-estradiol or PPT. Our results demonstrate a profound regulatory role of ERα in ryanodine receptor-dependent transition to chronic pain. PMID:27499186

  17. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  18. Presence and functional significance of presynaptic ryanodine receptors.

    PubMed

    Bouchard, Ron; Pattarini, Roberto; Geiger, Jonathan D

    2003-04-01

    Ca(2+)-induced Ca(2+) release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca(2+) concentration ([Ca(2+)](i)), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca(2+) to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca(2+)](i), (iii) how ER Ca(2+) load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca(2+) diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca(2+) stores are able to modulate local Ca(2+) levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function. PMID:12880633

  19. Ryanodine receptor dysfunction and triggered activity in the heart.

    PubMed

    Katra, Rodolphe P; Oya, Toshiyuki; Hoeker, Gregory S; Laurita, Kenneth R

    2007-05-01

    Arrhythmogenesis has been increasingly linked to cardiac ryanodine receptor (RyR) dysfunction. However, the mechanistic relationship between abnormal RyR function and arrhythmogenesis in the heart is not clear. We hypothesize that, under abnormal RyR conditions, triggered activity will be caused by spontaneous calcium release (SCR) events that depend on transmural heterogeneities of calcium handling. We performed high-resolution optical mapping of intracellular calcium and transmembrane potential in the canine left ventricular wedge preparation (n = 28). Rapid pacing was used to initiate triggered activity under normal and abnormal RyR conditions induced by FKBP12.6 dissociation and beta-adrenergic stimulation (20-150 microM rapamycin, 0.2 microM isoproterenol). Under abnormal RyR conditions, almost all preparations experienced SCRs and triggered activity, in contrast to control, rapamycin, or isoproterenol conditions alone. Furthermore, under abnormal RyR conditions, complex arrhythmias (monomorphic and polymorphic tachycardia) were commonly observed. After washout of rapamycin and isoproterenol, no triggered activity was observed. Surprisingly, triggered activity and SCRs occurred preferentially near the epicardium but not the endocardium (P < 0.01). Interestingly, the occurrence of triggered activity and SCR events could not be explained by cytoplasmic calcium levels, but rather by fast calcium reuptake kinetics. These data suggest that, under abnormal RyR conditions, triggered activity is caused by multiple SCR events that depend on the faster calcium reuptake kinetics near the epicardium. Furthermore, multiple regions of SCR may be a mechanism for multifocal arrhythmias associated with RyR dysfunction. PMID:17189349

  20. Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

    PubMed

    Diaz-Sylvester, Paula L; Porta, Maura; Copello, Julio A

    2011-01-01

    Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

  1. Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

    PubMed Central

    Diaz-Sylvester, Paula L.; Porta, Maura; Copello, Julio A.

    2011-01-01

    Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

  2. Ryanodine and inositol trisphosphate receptors are differentially distributed and expressed in rat parotid gland.

    PubMed Central

    Zhang, X; Wen, J; Bidasee, K R; Besch, H R; Wojcikiewicz, R J; Lee, B; Rubin, R P

    1999-01-01

    The present study examines the cellular distribution of the ryanodine receptor/channel (RyR) and inositol 1,4,5-trisphosphate receptor (InsP3R) subtypes in parotid acini. Using fluorescently labelled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid glycyl-ryanodine (BODIPYtrade mark-ryanodine) and confocal microscopy, RyRs were localized primarily to the perinuclear region (basal pole) of the acinar cell. Ryanodine, Ruthenium Red, cAMP and cADP ribose (cADPR) competed with BODIPY-ryanodine, resulting in a reduction in the fluorescence signal. However, inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3] did not alter the binding of BODIPY-ryanodine. Using receptor-subtype-specific antisera, InsP3Rs (types I, II and III) were located predominantly in the apical pole of the parotid cell. The presence of these three subtypes was confirmed using reverse transcriptase PCR with RNA-specific oligonucleotide probes. Binding studies using a parotid cell-membrane fraction identified and characterized RyRs and InsP3Rs in terms of binding affinity (Kd) and maximum binding capacity (Bmax) and confirmed that cADPR displaces ryanodine from its binding sites. Ruthenium Red and 8-Br-cADP-ribose blocked Ca2+ release in permeabilized acinar cells in response to cADPR and cAMP or forskolin, whereas Ins(1,4,5)P3-induced Ca2+ release was unaffected. The localization of the RyRs and InsP3Rs in discrete regions endow broad areas of the parotid cell with ligand-activated Ca2+ channels. The consequences of the dual activation of the RyRs and InsP3Rs by physiologically relevant stimuli such as noradrenaline (norepinephrine) are considered in relation to Ca2+ signalling in the parotid gland. PMID:10333498

  3. Disease Mutations in the Ryanodine Receptor Central Region: Crystal Structures of a Phosphorylation Hot Spot Domain

    SciTech Connect

    Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip

    2015-02-09

    Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain salt bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.

  4. Molecular characterization of a ryanodine receptor gene from Spodoptera exigua and its upregulation by chlorantraniliprole.

    PubMed

    Sun, Lina; Qiu, Guisheng; Cui, Li; Ma, Chunsen; Yuan, Huizhu

    2015-09-01

    Chlorantraniliprole is a novel diamide insecticide that targets the insect ryanodine receptor, a Ca(2+) release channel. Spodoptera exigua is a significant insect pest, and chlorantraniliprole is the most popular diamide insecticide used against this pest. To better understand the effects of diamides on RyR expression and [Ca(2+)], we isolated the SeRyR cDNA and investigated changes in SeRyR expression as a result of the application of chlorantraniliprole. The full-length cDNAs of SeRyR contain an open reading frame (ORF) of 15,357 bp with a predicted protein consisting of 5118 amino acids. SeRyR shares 77-92% identity with other insect RyR isoforms and 45-47% identity with vertebrate RyR isoforms. Furthermore, the relative expression abundances of RyR mRNA extracted from S. exigua fat body cells after 24 h of culture in 0.1, 1, 10, 100 nM, 1 µM and 100 µM of chlorantraniliprole changed 1.04-, 0.89-, 1.83-, 2.58-, 4.03- and 3.12-fold compared to blank control, respectively. The regression equation for the relative expression levels of SeRyR after 24 h as a function of the chlorantraniliprole concentration was Y = 0.6455 + 0.8188LgX, R(2) = 0.97093 for the cell line IOZCAS-Spex-II. These results outline the effects of chlorantraniliprole on the expression of SeRyR and provide a basis for the discovery of a compound that may exhibit selective insect activity. PMID:26267053

  5. Highly cooperative and hysteretic response of the skeletal muscle ryanodine receptor to changes in proton concentrations.

    PubMed Central

    Ma, J; Zhao, J

    1994-01-01

    Ryanodine receptors are key molecules in excitation-contraction coupling of skeletal muscle. They form the pore of the calcium release channel, which is regulated by Ca and ATP. Multiple proton titration sites are involved in controlling the different open states of the channel, as indicated by the following: i) the channel had a biphasic response to changes in proton concentrations around neutral pH; ii) the activities of the channel were inhibited by acidic pHs in a highly cooperative manner; and iii) the channel exhibited pronounced hysteresis to changes in pH. Four distinct conductance states can be identified in the single ryanodine-activated calcium release channel. The distribution of the multiple conductance states depends on the level of [Ca], ATP, and pH in the recording solution. The data are consistent with the multimeric structure of the skeletal muscle ryanodine receptor. Images FIGURE 3 PMID:7948677

  6. Spaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension.

    PubMed

    Dabertrand, Fabrice; Porte, Yves; Macrez, Nathalie; Morel, Jean-Luc

    2012-02-01

    Gravity has a structural role for living systems. Tissue development, architecture, and organization are modified when the gravity vector is changed. In particular, microgravity induces a redistribution of blood volume and thus pressure in the astronaut body, abolishing an upright blood pressure gradient, inducing orthostatic hypotension. The present study was designed to investigate whether isolated vascular smooth muscle cells are directly sensitive to altered gravitational forces and, second, whether sustained blood pressure changes act on the same molecular target. Exposure to microgravity during 8 days in the International Space Station induced the decrease of ryanodine receptor subtype 1 expression in primary cultured myocytes from rat hepatic portal vein. Identical results were found in portal vein from mice exposed to microgravity during an 8-day shuttle spaceflight. To evaluate the functional consequences of this physiological adaptation, we have compared evoked calcium signals obtained in myocytes from hindlimb unloaded rats, in which the shift of blood pressure mimics the one produced by the microgravity, with those obtained in myocytes from rats injected with antisense oligonucleotide directed against ryanodine receptor subtype 1. In both conditions, calcium signals implicating calcium-induced calcium release were significantly decreased. In contrast, in spontaneous hypertensive rat, an increase in ryanodine receptor subtype 1 expression was observed as well as the calcium-induced calcium release mechanism. Taken together, our results shown that myocytes were directly sensitive to gravity level and that they adapt their calcium signaling pathways to pressure by the regulation of the ryanodine receptor subtype 1 expression. PMID:22096120

  7. Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

    PubMed Central

    Hohenegger, Martin; Suko, Josef; Gscheidlinger, Regina; Drobny, Helmut; Zidar, Andreas

    2002-01-01

    Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel. PMID:12102654

  8. Rectification of muscle and nerve deficits in paralyzed ryanodine receptor type 1 mutant embryos

    PubMed Central

    Hanson, M. Gartz; Niswander, Lee A.

    2015-01-01

    Locomotion and respiration require motor axon connectivity and activation of the neuromuscular junction (NMJ). Through a forward genetic screen for muscle weakness, we recently reported an allele of ryanodine receptor type 1 (Ryr1AG). Here we reveal a role for functional RyR1 during acetylcholine receptor (AChR) cluster formation and embryonic synaptic transmission. Ryr1AG homozygous embryos are non-motile. Motor axons extend past AChR clusters and enlarged AChR clusters are found under fasciculated nerves. Using physiological and pharmacological methods, we show that contractility can be resumed through the masking of a potassium leak, and evoked vesicular release can be resumed via bypassing the defect in RyR1 induced calcium release. Moreover, we show the involvement of ryanodine receptors in presynaptic release at the NMJ. This data provides evidence of a role for RyR1 on both the pre- and postsynaptic sides of the NMJ. PMID:26025922

  9. Ryanodine receptors are involved in nuclear calcium oscillation in primary pancreatic {beta}-cells

    SciTech Connect

    Zheng, Ji; Chen, Zheng; Yin, Wenxuan; Miao, Lin; Zhou, Zhansong; Ji, Guangju

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Black-Right-Pointing-Pointer We showed that the pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. Black-Right-Pointing-Pointer Our results demonstrate that ryanodine-sensitive Ca{sup 2+} stores exist and have function in the pancreatic {beta}-cell nucleus. -- Abstract: Ryanodine receptors (RyRs) are mainly located on the endoplasmic reticulum (ER) and play an important role in regulating glucose-induced cytosolic Ca{sup 2+} oscillation in pancreatic {beta}-cells. However, subcellular locations and functions of RyRs on other cell organelles such as nuclear envelope are not well understood. In order to investigate the role of RyRs in nuclear Ca{sup 2+} oscillation we designed and conducted experiments in intact primary pancreatic {beta}-cells. Immunocytochemistry was used to examine the expression of RYRs on the nuclear envelope. Confocal microscopy was used to evaluate the function of RYRs on the nuclear envelope. We found that RyRs are expressed on the nuclear envelope in single primary pancreatic {beta}-cells and isolated nuclei. Laser scanning confocal microscopy studies indicated that application of glucose to the cells co-incubated with Ca{sup 2+} indicator Fluo-4 AM and cell-permeable nuclear indicator Hoechst 33342 resulted in nuclear Ca{sup 2+} oscillation. The pattern of glucose-induced Ca{sup 2+} oscillation in the nucleus and cytosol was similar. The reduction of Ca{sup 2+} oscillation amplitude by ryanodine was much greater in the nucleus though both the cytosol and the nucleus Ca{sup 2+} amplitude decreased by ryanodine. Our results suggest that functional ryanodine receptors not only exist in endoplasmic reticulum but are also expressed in nuclear envelope of pancreatic {beta}-cells.

  10. Function and expression of ryanodine receptors and inositol 1,4,5-trisphosphate receptors in smooth muscle cells of murine feed arteries and arterioles.

    PubMed

    Westcott, Erika B; Goodwin, Erica L; Segal, Steven S; Jackson, William F

    2012-04-15

    We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles. PMID:22331418

  11. The effects of compensated cardiac hypertrophy on dihydropyridine and ryanodine receptors in rat, ferret and guinea-pig hearts.

    PubMed

    Rannou, F; Sainte-Beuve, C; Oliviero, P; Do, E; Trouvé, P; Charlemagne, D

    1995-05-01

    The number of dihydropyridine and ryanodine receptors (DHP-R and RyR) has been measured in control and hypertrophied ventricles from rats, guinea pigs and ferrets to determine whether these two channels contribute to the alterations in excitation-contraction coupling (ECC), and in Ca2+ transient during compensated cardiac hypertrophy. We found that ventricular hypertrophy did not change the density of DHP-R. Mild hypertrophy did not alter the density of RyR in the rat but decreased it in the guinea-pig and in the ferret (30% and 36%, respectively). Severe hypertrophy decreased the density of RyR by 20% in the rat and by 34% in the guinea-pig. Therefore, the decrease is greater in ferret and guinea-pig hearts than in rat heart. We conclude that the sarcoplasmic reticulum (SR) Ca2+ release channels but not the L-type Ca2+ channels could contribute to the slowing of intracellular Ca2+ movements and to the reduced velocity of shortening of the hypertrophied hearts. We suggest that, in the guinea pig and ferret hearts which express only the beta myosin heavy chain (MHC) isoform, the reduced velocity of shortening during hypertrophy is related to the decrease in RyR density, whereas in the rat, it is regulated primarily via a shift in the MHC isoform, except in severe hypertrophy in which the moderate decrease in RyR would also be involved. PMID:7473781

  12. Glucose-Dependent Insulin Secretion in Pancreatic β-Cell Islets from Male Rats Requires Ca2+ Release via ROS-Stimulated Ryanodine Receptors

    PubMed Central

    Llanos, Paola; Contreras-Ferrat, Ariel; Barrientos, Genaro; Valencia, Marco; Mears, David; Hidalgo, Cecilia

    2015-01-01

    Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells requires an increase in intracellular free Ca2+ concentration ([Ca2+]). Glucose uptake into β-cells promotes Ca2+ influx and reactive oxygen species (ROS) generation. In other cell types, Ca2+ and ROS jointly induce Ca2+ release mediated by ryanodine receptor (RyR) channels. Therefore, we explored here if RyR-mediated Ca2+ release contributes to GSIS in β-cell islets isolated from male rats. Stimulatory glucose increased islet insulin secretion, and promoted ROS generation in islets and dissociated β-cells. Conventional PCR assays and immunostaining confirmed that β-cells express RyR2, the cardiac RyR isoform. Extended incubation of β-cell islets with inhibitory ryanodine suppressed GSIS; so did the antioxidant N-acetyl cysteine (NAC), which also decreased insulin secretion induced by glucose plus caffeine. Inhibitory ryanodine or NAC did not affect insulin secretion induced by glucose plus carbachol, which engages inositol 1,4,5-trisphosphate receptors. Incubation of islets with H2O2 in basal glucose increased insulin secretion 2-fold. Inhibitory ryanodine significantly decreased H2O2-stimulated insulin secretion and prevented the 4.5-fold increase of cytoplasmic [Ca2+] produced by incubation of dissociated β-cells with H2O2. Addition of stimulatory glucose or H2O2 (in basal glucose) to β-cells disaggregated from islets increased RyR2 S-glutathionylation to similar levels, measured by a proximity ligation assay; in contrast, NAC significantly reduced the RyR2 S-glutathionylation increase produced by stimulatory glucose. We propose that RyR2-mediated Ca2+ release, induced by the concomitant increases in [Ca2+] and ROS produced by stimulatory glucose, is an essential step in GSIS. PMID:26046640

  13. Evidence for the transport of glutathione through ryanodine receptor channel type 1.

    PubMed Central

    Bánhegyi, Gábor; Csala, Miklós; Nagy, Gábor; Sorrentino, Vincenzo; Fulceri, Rosella; Benedetti, Angelo

    2003-01-01

    In the present study, we have investigated the role of RyR1 (ryanodine receptor calcium channel type 1) in glutathione (GSH) transport through the sarcoplasmic reticulum (SR) membrane of skeletal muscles. Lanthanum chloride, a prototypic blocker of cation channels, inhibited the influx and efflux of GSH in SR vesicles. Using a rapid-filtration-based assay and lanthanum chloride as a transport blocker, an uptake of radiolabelled GSH into SR vesicles was observed. Pretreatment of SR vesicles with the RyR1 antagonists Ruthenium Red and ryanodine as well as with lanthanum chloride blocked the GSH uptake. An SR-like GSH uptake appeared in microsomes obtained from an HEK-293 (human embryonic kidney 293) cell line after transfection of RyR1. These observations strongly suggest that RyR1 mediates GSH transport through the SR membranes of skeletal muscles. PMID:14519101

  14. Dust from hog confinement facilities impairs Ca2+ mobilization from sarco(endo)plasmic reticulum by inhibiting ryanodine receptors

    PubMed Central

    Tian, Chengju; Moore, Caronda J.; Dodmane, Puttappa; Shao, Chun Hong; Romberger, Debra J.; Toews, Myron L.

    2013-01-01

    Individuals working in commercial hog confinement facilities have elevated incidences of headaches, depression, nausea, skeletal muscle weakness, fatigue, gastrointestinal disorders, and cardiovascular diseases, and the molecular mechanisms for these nonrespiratory ailments remain incompletely undefined. A common element underlying these diverse pathophysiologies is perturbation of intracellular Ca2+ homeostasis. This study assessed whether the dust generated inside hog confinement facilities contains compounds that alter Ca2+ mobilization via ryanodine receptors (RyRs), key intracellular channels responsible for mobilizing Ca2+ from internal stores to elicit an array of physiologic functions. Hog barn dust (HBD) was extracted with phosphate-buffered saline, sterile-filtered (0.22 μm), and size-separated using Sephadex G-100 resin. Fractions (F) 1 through 9 (Mw >10,000 Da) had no measurable effects on RyR isoforms. However, F10 through F17, which contained compounds of Mw ≤2,000 Da, modulated the [3H]ryanodine binding to RyR1, RyR2, and RyR3 in a biphasic (Gaussian) manner. The Ki values for F13, the most potent fraction, were 3.8 ± 0.2 μg/ml for RyR1, 0.2 ± 0.01 μg/ml and 19.1 ± 2.8 μg/ml for RyR2 (two binding sites), and 44.9 ± 2.8 μg/ml and 501.6 ± 9.2 μg/ml for RyR3 (two binding sites). In lipid bilayer assays, F13 dose-dependently decreased the open probabilities of RyR1, RyR2, and RyR3. Pretreating differentiated mouse skeletal myotubes (C2C12 cells) with F13 blunted the amplitudes of ryanodine- and K+-induced Ca2+ transients. Because RyRs are present in many cell types, impairment in Ca2+ mobilization from internal stores via these channels is a possible mechanism by which HBD may trigger these seemingly unrelated pathophysiologies. PMID:23288552

  15. Structure-activity relationship of non-coplanar polychlorinated biphenyls toward skeletal muscle ryanodine receptors in rainbow trout (Oncorhynchus mykiss)

    PubMed Central

    Fritsch, Erika B.; Pessah, Isaac N.

    2013-01-01

    Research addressing the health impacts of polychlorinated biphenyls (PCBs) has primarily focused on the effects of coplanar, or dioxin-like (DL), congeners, which is especially true for research assessing impacts in fish species. Ortho substituted non-coplanar, termed non-dioxin-like (NDL), PCBs have received less attention. In mammals, NDL PCBs enhance the activity of ryanodine receptors (RyR), calcium release channels necessary for engaging excitation-contraction (EC) coupling in striated muscle. We utilized in vitro receptor binding analysis to determine whether NDL PCB congeners detected in aquatic environments alter the activity of RyR isoform 1 (RyR1) found in the skeletal muscle of rainbow trout. Congeners 52, 95, 136, and 149 were the most efficacious leading to an increase in receptor activity that was approximately 250% greater than that found under solvent control conditions. Other environmentally relevant congeners, namely PCB 153, 151 and 101, which all contain two or more chlorines in the ortho-position, enhanced receptor activity by greater than 160% of baseline. The mono-ortho congeners or the non-ortho PCB 77 had negligible impact on the RyR1. When combined, in binary or environmentally relevant mixtures, congeners shown to enhance receptor activity appeared to display additivity and when the active PCB 95 was present with the non-active congener PCB 77 the impact on receptor activity was reduced from 250% to 230%. The important role of the RyR and the demonstrated additive nature of NDL congeners towards altering channel function calls for further investigation into the ecological implications of altered RyR function in fish with high PCB burdens. PMID:23827775

  16. Carbonylation Induces Heterogeneity in Cardiac Ryanodine Receptor Function in Diabetes Mellitus

    PubMed Central

    Shao, Chun Hong; Tian, Chengju; Ouyang, Shouqiang; Moore, Caronda J.; Alomar, Fadhel; Nemet, Ina; D'Souza, Alicia; Nagai, Ryoji; Kutty, Shelby; Rozanski, George J.; Ramanadham, Sasanka; Singh, Jaipaul

    2012-01-01

    Heart failure and arrhythmias occur at 3 to 5 times higher rates among individuals with diabetes mellitus, compared with age-matched, healthy individuals. Studies attribute these defects in part to alterations in the function of cardiac type 2 ryanodine receptors (RyR2s), the principal Ca2+-release channels on the internal sarcoplasmic reticulum (SR). To date, mechanisms underlying RyR2 dysregulation in diabetes remain poorly defined. A rat model of type 1 diabetes, in combination with echocardiography, in vivo and ex vivo hemodynamic studies, confocal microscopy, Western blotting, mass spectrometry, site-directed mutagenesis, and [3H]ryanodine binding, lipid bilayer, and transfection assays, was used to determine whether post-translational modification by reactive carbonyl species (RCS) represented a contributing cause. After 8 weeks of diabetes, spontaneous Ca2+ release in ventricular myocytes increased ∼5-fold. Evoked Ca2+ release from the SR was nonuniform (dyssynchronous). Total RyR2 protein levels remained unchanged, but the ability to bind the Ca2+-dependent ligand [3H]ryanodine was significantly reduced. Western blotting and mass spectrometry revealed RCS adducts on select basic residues. Mutation of residues to delineate the physiochemical impact of carbonylation yielded channels with enhanced or reduced cytoplasmic Ca2+ responsiveness. The prototype RCS methylglyoxal increased and then decreased the RyR2 open probability. Methylglyoxal also increased spontaneous Ca2+ release and induced Ca2+ waves in healthy myocytes. Treatment of diabetic rats with RCS scavengers normalized spontaneous and evoked Ca2+ release from the SR, reduced carbonylation of RyR2s, and increased binding of [3H]ryanodine to RyR2s. From these data, we conclude that post-translational modification by RCS contributes to the heterogeneity in RyR2 activity that is seen in experimental diabetes. PMID:22648972

  17. The role of ryanodine receptor type 3 in a mouse model of Alzheimer disease

    PubMed Central

    Liu, Jie; Supnet, Charlene; Sun, Suya; Zhang, Hua; Good, Levi; Popugaeva, Elena; Bezprozvanny, Ilya

    2014-01-01

    Dysregulated endoplasmic reticulum (ER) calcium (Ca2+) signaling is reported to play an important role in Alzheimer disease (AD) pathogenesis. The role of ER Ca2+ release channels, the ryanodine receptors (RyanRs), has been extensively studied in AD models and RyanR expression and activity are upregulated in the brains of various familial AD (FAD) models. The objective of this study was to utilize a genetic approach to evaluate the importance of RyanR type 3 (RyanR3) in the context of AD pathology. PMID:24476841

  18. A rare genetic variant of the ryanodine receptor in a suspected malignant hyperthermia susceptible patient.

    PubMed

    MacKay, Emily Jane; Wilkerson, Carlos; Kraeva, Natalia; Rosenberg, Henry; Kennedy, Tara

    2016-09-01

    Malignant hyperthermia (MH) remains a diagnostic challenge. This case report describes the anesthetic management of a suspected intraoperative MH episode and the subsequent, genetic sequence analysis of 3 genes associated with MH. The results of the molecular genetic testing revealed heterozygosity for a rare variant, c.12553G>A (p.Ala4185Thr), in the RYR1 gene encoding the ryanodine receptor. Although the RYR1 gene has previously been implicated in the pathogenesis of MH, (1) this particular variant has only been reported in one other case of MH; (2) the role for diagnostic genetic testing in the diagnosis of MH will be examined. PMID:27555149

  19. FKBP12.6 binding of ryanodine receptors carrying mutations associated with arrhythmogenic cardiac disease.

    PubMed

    Zissimopoulos, Spyros; Thomas, N Lowri; Jamaluddin, Wan W; Lai, F Anthony

    2009-04-15

    In the present paper we show that distinct human RyR2 (ryanodine receptor type 2) inherited mutations expressed in mammalian cells exhibit either unaltered or increased FKBP12.6 (12.6 kDa FK506-binding protein) binding compared with the wild-type. Oxidizing conditions result in decreased FKBP12.6 binding, but to the same extent as for the wild-type. Our findings suggest that FKBP12.6 regulation of RyR2 is unlikely to be the primary defect in inherited arrhythmogenic cardiac disease. PMID:19226252

  20. Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface

    PubMed Central

    Kimlicka, Lynn; Lau, Kelvin; Tung, Ching-Chieh; Van Petegem, Filip

    2013-01-01

    Ryanodine receptors are large channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of RyR mutations can cause cardiac and skeletal muscle disorders, yet detailed mechanisms explaining their effects have been lacking. Here we compare pseudo-atomic models and propose that channel opening coincides with widening of a cytoplasmic vestibule formed by the N-terminal region, thus altering an interface targeted by 20 disease mutations. We solve crystal structures of several disease mutants that affect intrasubunit domain–domain interfaces. Mutations affecting intrasubunit ionic pairs alter relative domain orientations, and thus couple to surrounding interfaces. Buried disease mutations cause structural changes that also connect to the intersubunit contact area. These results suggest that the intersubunit contact region between N-terminal domains is a prime target for disease mutations, direct or indirect, and we present a model whereby ryanodine receptors and inositol-1,4,5-trisphosphate receptors are activated by altering domain arrangements in the N-terminal region. PMID:23422674

  1. Ryanodine receptors selectively contribute to the formation of taste-evoked calcium signals in mouse taste cells

    PubMed Central

    Rebello, Michelle R.; Medler, Kathryn F.

    2010-01-01

    The peripheral taste system uses multiple signaling pathways to transduce a stimulus into an output signal that activates afferent neurons. All of these signaling pathways depend on transient increases in intracellular calcium but the current understanding of these calcium signals is not well-developed. Using molecular and physiological techniques, this study establishes that ryanodine receptors (RyRs), specifically isoform 1, are expressed in taste cells and that their physiological function differs among cell types employing different signaling pathways. RyR1 contributes to some taste-evoked signals that rely on calcium release from internal stores but can also supplement the calcium signal that is initiated by opening VGCCs. In taste cells expressing both signaling pathways, RyR1 contributes to the depolarization-induced calcium signal but not to the calcium signal that depends on calcium release from stores. These data suggest that RyR1 is an important regulator of calcium signaling and that its physiological role in taste cells is dictated by the nature of the calcium signaling mechanisms expressed. PMID:20955474

  2. Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

    PubMed Central

    Westcott, Erika B.

    2011-01-01

    The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP3Rs in Ca2+ signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca2+ sparks and Ca2+ waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca2+ and constricted the arteries. The blockade of IP3Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca2+ waves without affecting Ca2+ sparks. Importantly, the IP3Rs and phospholipase C antagonists decreased global intracellular Ca2+ and dilated the arteries. In contrast, cremaster arterioles displayed only Ca2+ waves: Ca2+ sparks were not observed, and neither ryanodine (10–50 μM) nor tetracaine (100 μM) affected either Ca2+ signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca2+ waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca2+ and vasodilation. These findings highlight the contrasting roles played by RyRs and IP3Rs in Ca2+ signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles. PMID:21357503

  3. S100A1 and Calmodulin Compete for the Same Binding Site on Ryanodine Receptor*

    PubMed Central

    Wright, Nathan T.; Prosser, Benjamin L.; Varney, Kristen M.; Zimmer, Danna B.; Schneider, Martin F.; Weber, David J.

    2008-01-01

    In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca2+ release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca2+-S100A1 competes with Ca2+-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca2+-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca2+-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca2+-CaM. A model for regulating muscle contraction is presented in which Ca2+-S100A1 and Ca2+-CaM compete directly for the same binding site on the ryanodine receptor. PMID:18650434

  4. Ca(2+) microdomains, NAADP and type 1 ryanodine receptor in cell activation.

    PubMed

    Guse, Andreas H; Wolf, Insa M A

    2016-06-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) mobilizing second messenger that belongs to the superfamily of regulatory adenine nucleotides. Though NAADP has been known since 20years, several aspects of its metabolism and molecular mode of action are still under discussion. Though the importance of the type 1 ryanodine receptor was discovered and published already in 2002 Hohenegger et al. (2002 Oct 15) , recent data re-emphasize these original findings in pancreatic acinar cells and in T-lymphocytes. Here we review recent developments in NAADP formation and metabolism, putative target Ca(2+) channels for NAADP with special emphasis on the type 1 ryanodine receptor, and NAADP binding proteins. The latter are basis for a unifying hypothesis for NAADP action. Finally, the role of NAADP in T cell Ca(2+) signaling and activation is discussed. This article is part of a Special Issue entitled: Calcium and Cell Fate . Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26804481

  5. Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

    PubMed

    Xiao, Liang; Gurrola, Georgina B; Zhang, Jing; Valdivia, Carmen R; SanMartin, Mario; Zamudio, Fernando Z; Zhang, Liming; Possani, Lourival D; Valdivia, Héctor H

    2016-05-01

    Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin > urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency. PMID:27114612

  6. Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor.

    PubMed

    Mei, Yingwu; Xu, Le; Mowrey, David D; Mendez Giraldez, Raul; Wang, Ying; Pasek, Daniel A; Dokholyan, Nikolay V; Meissner, Gerhard

    2015-07-10

    Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes. PMID:25998124

  7. The mitochondrial ryanodine receptor in rat heart: a pharmaco-kinetic profile.

    PubMed

    Altschafl, Beth A; Beutner, Gisela; Sharma, Virendra K; Sheu, Shey-Shing; Valdivia, Héctor H

    2007-07-01

    A protein discovered within inner mitochondrial membranes (IMM), designated as the mitochondrial ryanodine receptor (mRyR), has been recognized recently as a modulator of Ca(2+) fluxes in mitochondria. The present study provides fundamental pharmacological and electrophysiological properties of this mRyR. Rat cardiac IMM fused to lipid bilayers revealed the presence of a mitochondrial channel with gating characteristics similar to those of classical sarcoplasmic reticulum RyR (SR-RyR), but a variety of other mitochondrial channels obstructed clean recordings. Mitochondrial vesicles were thus solubilized and subjected to sucrose sedimentation to obtain mRyR-enriched fractions. Reconstitution of sucrose-purified fractions into lipid bilayers yielded Cs(+)-conducting, Ca(2+)-sensitive, large conductance (500-800 pS) channels with signature properties of SR-RyRs. Cytosolic Ca(2+) increased the bursting frequency and mean open time of the channel. Micromolar concentrations of ryanodine induced the appearance of subconductance states or inhibited channel activity altogether, while Imperatoxin A (IpTx(a)), a specific activator of RyRs, reversibly induced the appearance of distinct subconductance states. Remarkably, the cardiac mRyR displayed a Ca(2+) dependence of [(3)H]ryanodine binding curve similar to skeletal RyR (RyR1), not cardiac RyR (RyR2). Overall, the mRyR displayed elemental attributes that are present in single channel lipid bilayer recordings of SR-RyRs, although some exquisite differences were also noted. These results therefore provide the first direct evidence that a unique RyR occurs in mitochondrial membranes. PMID:17499575

  8. Involvement of nitric oxide in iodine deficiency-induced microvascular remodeling in the thyroid gland: role of nitric oxide synthase 3 and ryanodine receptors.

    PubMed

    Craps, J; Wilvers, C; Joris, V; De Jongh, B; Vanderstraeten, J; Lobysheva, I; Balligand, J-L; Sonveaux, P; Gilon, P; Many, M-C; Gérard, A-C; Colin, I M

    2015-02-01

    Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in 3 in vitro models of ID, including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitroarginine methyl ester (a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, whereas S-nitroso-acetyl-penicillamine (a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the 3 known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10μM decreased ID-induced NOS3 activation, HIF-1α, and VEGF-A expression, whereas RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes. PMID:25406019

  9. Expression of Dihydropyridine and Ryanodine Receptors in Type IIA Fibers of Rat Skeletal Muscle

    PubMed Central

    Anttila, Katja; Mänttäri, Satu; Järvilehto, Matti

    2007-01-01

    In this study, the fiber type specificity of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs) in different rat limb muscles was investigated. Western blot and histochemical analyses provided for the first time evidence that the expression of both receptors correlates to a specific myosin heavy chain (MHC) composition. We observed a significant (p=0.01) correlation between DHP as well as Ry receptor density and the expression of MHC IIa (correlation factor r=0.674 and r=0.645, respectively) in one slow-twitch, postural muscle (m. soleus), one mixed, fast-twitch muscle (m. gastrocnemius) and two fast-twitch muscles (m. rectus femoris, m. extensor digitorum longus). The highest DHP and Ry receptor density was found in the white part of m. rectus femoris (0.058±0.0060 and 0.057±0.0158 ODu, respectively). As expected, the highest relative percentage of MHC IIa was also found in the white part of m. rectus femoris (70.0±7.77%). Furthermore, histochemical experiments revealed that the IIA fibers stained most strongly for the fluorophore-conjugated receptor blockers. Our data clearly suggest that the expression of DHPRs and RyRs follows a fiber type-specific pattern, indicating an important role for these proteins in the maintenance of an effective Ca2+ cycle in the fast contracting fiber type IIA. PMID:17576431

  10. Reactive oxygen species-mediated neurodegeneration is independent of the ryanodine receptor in Caernorhabditis elegans

    PubMed Central

    Young, Lyndsay EA; Williams, Daniel C.

    2016-01-01

    Despite the significant impacts on human health caused by neurodegeneration, our understanding of the degeneration process is incomplete. The nematode Caenorhabditis elegans is emerging as a genetic model organism well suited for identification of conserved cellular mechanisms and molecular pathways of neurodegeneration. Studies in the worm have identified factors that contribute to neurodegeneration, including excitotoxicity and stress due to reactive oxygen species (ROS). Disruption of the gene unc-68, which encodes the ryanodine receptor, abolishes excitotoxic cell death, indicating a role for calcium (Ca2+) signaling in neurodegeneration. We tested the requirement for unc-68 in ROS-mediated neurodegeneration using the genetically encoded photosensitizer KillerRed. Upon illumination of KillerRed expressing animals to produce ROS, we observed similar levels of degeneration in wild-type and unc-68 mutant strains. Our results indicate that ROS-mediated cell death is independent of unc-68 and suggest multiple molecular pathways of neurodegeneration.

  11. Selecting Ions by Size in a Calcium Channel: The Ryanodine Receptor Case Study

    PubMed Central

    Gillespie, Dirk; Xu, Le; Meissner, Gerhard

    2014-01-01

    Many calcium channels can distinguish between ions of the same charge but different size. For example, when cations are in direct competition with each other, the ryanodine receptor (RyR) calcium channel preferentially conducts smaller cations such as Li+ and Na+ over larger ones such as K+ and Cs+. Here, we analyze the physical basis for this preference using a previously established model of RyR permeation and selectivity. Like other calcium channels, RyR has four aspartate residues in its GGGIGDE selectivity filter. These aspartates have their terminal carboxyl group in the pore lumen, which take up much of the available space for permeating ions. We find that small ions are preferred by RyR because they can fit into this crowded environment more easily. PMID:25418295

  12. Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles.

    PubMed

    Westcott, Erika B; Jackson, William F

    2011-05-01

    The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(²+) sparks and Ca(²+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(²+) and constricted the arteries. The blockade of IP₃Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(²+) sparks. Importantly, the IP₃Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(²+) waves: Ca(²+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(²+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(²+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(²+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP₃Rs in Ca(²+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles. PMID:21357503

  13. Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor.

    PubMed Central

    Bhat, M B; Zhao, J; Takeshima, H; Ma, J

    1997-01-01

    The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:9284301

  14. Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

    PubMed Central

    Kobayashi, Shigeki; Yamamoto, Takeshi; Parness, Jerome; Ikemoto, Noriaki

    2004-01-01

    N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function. PMID:15027895

  15. Novel Anthranilic Diamide Scaffolds Containing N-Substituted Phenylpyrazole as Potential Ryanodine Receptor Activators.

    PubMed

    Liu, Jing-Bo; Li, Yu-Xin; Zhang, Xiu-Lan; Hua, Xue-Wen; Wu, Chang-Chun; Wei, Wei; Wan, Ying-Ying; Cheng, Dan-Dan; Xiong, Li-Xia; Yang, Na; Song, Hai-Bin; Li, Zheng-Ming

    2016-05-11

    To discover potent insecticides targeting ryanodine receptors (RyRs), a series of novel anthranilic diamides analogues (12a-12u) containing N-substituted phenylpyrazole were designed and synthesized. These compounds were characterized by (1)H NMR, (13)C NMR, and HRMS, and the structure of compound 12u was confirmed by X-ray diffraction. Their insecticidal activities indicated that these compounds displayed moderate to excellent activities. In particular, 12i showed 100 and 37% larvicidal activities against oriental armyworm (Mythimna separata) at 0.25 and 0.05 mg L(-1), equivalent to that of chlorantraniliprole (100%, 0.25 mg L(-1); and 33%, 0.05 mg L(-1)). The activity of 12i against diamondback moth (Plutella xylostella) was 95% at 0.05 mg L(-1), whereas the control was 100% at 0.05 mg L(-1). The calcium-imaging technique experiment results showed that the effects of 12i on the intracellular calcium ion concentration ([Ca(2+)]i) in neurons were concentration-dependent. After the central neurons of Helicoverpa armigera were dyed by loading with fluo-5N and treated with 12i, the free calcium released in endoplasmic reticulum indicated the target of compound 12i is RyRs or IP3Rs. The activation of RyRs by natural ryanodine completely blocked the calcium release induced by 12i, which indicated that RyRs in the central neurons of H. armigera third-instar larvae is the possible target of compound 12i. PMID:27109555

  16. Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

    PubMed Central

    Chen, S R; Leong, P; Imredy, J P; Bartlett, C; Zhang, L; MacLennan, D H

    1997-01-01

    We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel. Images FIGURE 1 PMID:9336186

  17. Complex formation between calsequestrin and the ryanodine receptor in fast- and slow-twitch rabbit skeletal muscle.

    PubMed

    Murray, B E; Ohlendieck, K

    1998-06-16

    Linkage between the high-capacity Ca2+-binding protein calsequestrin and the ryanodine receptor is proposed to be essential for proper Ca2+-release during skeletal muscle excitation-contraction coupling. However, no direct biochemical evidence exists showing a connection between these high-molecular-mass complexes in native skeletal muscle membranes. Here, using immunoblot analysis of chemically crosslinked membrane vesicles enriched in triad junctions, we have demonstrated that a very close neighborhood relationship exists between calsequestrin and the ryanodine receptor in both main fiber types. Hence, the luminal Ca2+-reservoir complex appears to be directly coupled to the membrane Ca2+-release complex and oligomerization seems to be of functional importance. PMID:9662440

  18. Minding the Calcium Store: Ryanodine Receptor Activation as a Convergent Mechanism of PCB Toxicity

    PubMed Central

    Pessah, Isaac N.; Cherednichenko, Gennady; Lein, Pamela J.

    2009-01-01

    Chronic low level polychlorinated biphenyls (PCB) exposures remain a significant public health concern since results from epidemiological studies indicate PCB burden is associated with immune system dysfunction, cardiovascular disease, and impairment of the developing nervous system. Of these various adverse health effects, developmental neurotoxicity has emerged as a particularly vulnerable endpoint in PCB toxicity. Arguably the most pervasive biological effects of PCBs could be mediated by their ability to alter the spatial and temporal fidelity of Ca2+ signals through one or more receptor mediated processes. This review will focus on our current knowledge of the structure and function of ryanodine receptors (RyRs) in muscle and nerve cells and how PCBs and related non-coplanar structures alter these functions. The molecular and cellular mechanisms by which non-coplanar PCBs and related structures alter local and global Ca2+ signaling properties and the possible short and long-term consequences of these perturbations on neurodevelopment and neurodegeneration are reviewed. PMID:19931307

  19. Effect of ruthenium red, a ryanodine receptor antagonist in experimental diabetes induced vascular endothelial dysfunction and associated dementia in rats.

    PubMed

    Jain, Swati; Sharma, Bhupesh

    2016-10-01

    Diabetes mellitus is considered as a main risk factor for vascular dementia. In the past, we have reported the induction of vascular dementia by experimental diabetes. This study investigates the efficacy of a ruthenium red, a ryanodine receptor antagonist and pioglitazone in the pharmacological interdiction of pancreatectomy diabetes (PaD) induced vascular endothelial dysfunction and subsequent vascular dementia in rats. Attentional set shifting and Morris water-maze test were used for assessment of learning and memory. Vascular endothelial function, blood brain barrier permeability, serum glucose, serum nitrite/nitrate, oxidative stress (viz. aortic superoxide anion, brain thiobarbituric acid reactive species and brain glutathione), brain calcium and inflammation (myeloperoxidase) were also estimated. PaD rats have shown impairment of endothelial function, blood brain barrier permeability, learning and memory along with an increase in brain inflammation, oxidative stress and calcium. Administration of ruthenium red and pioglitazone has significantly attenuated PaD induced impairment of learning, memory, blood brain barrier permeability, endothelial function and biochemical parameters. It may be concluded that ruthenium red, a ryanodine receptor antagonist and pioglitazone, a PPAR-γ agonist may be considered as potent pharmacological agent for the management of PaD induced endothelial dysfunction and subsequent vascular dementia. Ryanodine receptor may be explored further for their possible benefits in vascular dementia. PMID:27262216

  20. IL-6/STAT3 signaling in mice with dysfunctional type-2 ryanodine receptor

    PubMed Central

    Huang, Tai-Qin; Willis, Monte S; Meissner, Gerhard

    2015-01-01

    ABSTRACT Mice with genetically modified cardiac ryanodine receptor (Ryr2ADA/ADA mice) are impaired in regulation by calmodulin, develop severe cardiac hypertrophy and die about 2 weeks after birth. We hypothesized that the interleukin 6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling pathway has a role in the development of the Ryr2ADA/ADA cardiac hypertrophy phenotype, and determined cardiac function and protein levels of IL-6, phosphorylation levels of STAT3, and downstream targets c-Fos and c-Myc in wild-type and RyR2ADA/ADA mice, mice with a disrupted IL-6 gene, and mice treated with STAT3 inhibitor NSC74859. IL-6 protein levels were increased at postnatal day 1 but not day 10, whereas pSTAT3-Tyr705/STAT3 ratio and c-Fos and c-Myc protein levels increased in hearts of 10-day but not 1-day old Ryr2ADA/ADA mice compared with wild type. Both STAT3 and pSTAT3-Tyr705 accumulated in the nuclear fraction of 10-day old Ryr2ADA/ADA mice compared with wild type. Ryr2ADA /ADA /IL-6−/− mice lived 1.5 times longer, had decreased heart to body weight ratio, and reduced c-Fos and c-Myc protein levels. The STAT3 inhibitor NSC74859 prolonged life span by 1.3-fold, decreased heart to body weight ratio, increased cardiac performance, and decreased pSTAT-Tyr705/STAT3 ratio and IL-6, c-Fos and c-Myc protein levels of Ryr2ADA /ADA mice. The results suggest that upregulation of IL-6 and STAT3 signaling contributes to cardiac hypertrophy and early death of mice with a dysfunctional ryanodine receptor. They further suggest that STAT3 inhibitors may be clinically useful agents in patients with altered Ca2+ handling in the heart. PMID:27217982

  1. FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

    PubMed Central

    O'Reilly, Fiona M; Robert, Mylène; Jona, Istvan; Szegedi, Csaba; Albrieux, Mireille; Geib, Sandrine; De Waard, Michel; Villaz, Michel; Ronjat, Michel

    2002-01-01

    In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) antagonizes this effect. Two peptides, covering these sequences (peptide A(Sk) and C(Sk), respectively) were immobilized on polystyrene beads. We demonstrate that peptide A(Sk) binds to the skeletal isoform of RyR (RyR1) whereas peptide C(Sk) does not. Using surface plasmon resonance detection, we show that 1) domain Thr(671)-Leu(690) is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A(Sk) with RyR1 is not modulated by Ca(2+) (pCa 9-2) nor by Mg(2+) (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC(50) = 10 nM) and inhibited by both rapamycin (IC(50) = 5 nM) and FK506. Peptide A(Sk) induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A(Sk) on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR alpha(1)-subunit corresponding to Thr(671)-Leu(690) and show that the association of FKBP12 with RyR1 specifically modulates this interaction. PMID:11751303

  2. Structure of the rabbit ryanodine receptor RyR1 at near-atomic resolution

    PubMed Central

    Wu, Jianping; Li, Zhangqiang; Xie, Tian; Peng, Wei; Yin, Changcheng; Li, Xueming; Scheres, Sjors H.W.; Shi, Yigong; Yan, Nieng

    2014-01-01

    The ryanodine receptors (RyRs) are high-conductance intracellular Ca2+ channels that play a pivotal role in the excitation-contraction coupling of skeletal and cardiac muscles. RyRs are the largest known ion channels, with a homotetrameric organization and approximately 5000 residues in each protomer. Here we report the structure of the rabbit RyR1 in complex with its modulator FKBP12 at an overall resolution of 3.8 Å, determined by single-particle electron cryo-microscopy. Three previously uncharacterized domains, named Central, Handle, and Helical domains, display the armadillo repeat fold. These domains, together with the amino-terminal domain, constitute a network of superhelical scaffold for binding and propagation of conformational changes. The channel domain exhibits the voltage-gated ion channel superfamily fold with distinct features. A negative charge-enriched hairpin loop connecting S5 and the pore helix is positioned above the entrance to the selectivity filter vestibule. The four elongated S6 segments form a right-handed helical bundle that closes the pore at the cytoplasmic border of the membrane. Allosteric regulation of the pore by the cytoplasmic domains is mediated through extensive interactions between the Central domains and the channel domain. These structural features explain high ion conductance by RyRs and the long-range allosteric regulation of channel activities. PMID:25517095

  3. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca(2+) release channel.

    PubMed

    Filipova, Dilyana; Walter, Anna M; Gaspar, John A; Brunn, Anna; Linde, Nina F; Ardestani, Mostafa A; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-01-01

    In mature skeletal muscle, the intracellular Ca(2+) concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca(2+) release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1's potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca(2+) signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca(2+) signaling during muscle organ development. PMID:26831464

  4. Depletion of FKBP does not affect the interaction between isolated ryanodine receptors

    SciTech Connect

    Hu Xiaofang; Liang Xin; Chen Keying; Zhu Peihong; Hu Jun; E-mail: jhu@sjtu.edu.cn

    2005-10-14

    The ryanodine receptors/calcium release channels (RyRs) usually form two dimensional regular lattices in the endoplasmic/sarcoplasmic reticulum membranes. The native RyR is associated with many auxiliary proteins, including FKBP. It has been indicated that FKBP may play a role in the intermolecular interaction and coupled gating of neighboring RyRs. However, a more recent study shows that FKBP12 is not involved in the physical linkage between neighboring RyR1s. In the present work, the effect of FKBP12 on the interaction between RyR1s isolated from rabbit skeletal muscle was investigated in an aqueous medium with photon correlation spectroscopy. We found that the depletion of FKBP12 did not affect the oligomerization of RyR1s in the medium containing different [KCl] or under different channel functional states. No evidence is obtained for the involvement of FKBP12 in the intermolecular interaction between RyR1s.

  5. Temperature acclimation has no effect on ryanodine receptor expression or subcellular localization in rainbow trout heart.

    PubMed

    Birkedal, Rikke; Christopher, Jennifer; Thistlethwaite, Angela; Shiels, Holly A

    2009-11-01

    In cardiomyocytes, ryanodine receptors (RYRs) mediate Ca(2+)-induced Ca(2+)-release (CICR) from the sarcoplasmic reticulum (SR) during excitation-contraction (e-c) coupling. In rainbow trout heart, the relative importance of CICR increases with cold-acclimation. Thus, the aim of this study was to investigate the effect of temperature acclimation (4, 11 and 18 degrees C) on RYR intracellular localization and expression density. We used immunocytochemistry to assess intracellular localization in ventricular myocytes and Western blotting to assess RYR expression in both atrial and ventricular tissue. In ventricular myocytes, RYRs were localized peripherally in transverse bands aligning with sarcomeric m-lines and centrally around mitochondria and the nucleus. Localization did not change with temperature acclimation. RYR expression was also unaffected by temperature acclimation. The localization of RYRs at the m-line is similar to neonatal mammalian cardiomyocytes. We suggest this positioning is indicative of myocytes which rely predominantly on transsarcolemmal Ca(2+)-influx, rather than CICR, during e-c coupling. PMID:19544062

  6. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca2+ release channel

    PubMed Central

    Filipova, Dilyana; Walter, Anna M.; Gaspar, John A.; Brunn, Anna; Linde, Nina F.; Ardestani, Mostafa A.; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-01-01

    In mature skeletal muscle, the intracellular Ca2+ concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca2+ release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1’s potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca2+ signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca2+ signaling during muscle organ development. PMID:26831464

  7. Posttranslational modifications of cardiac ryanodine receptors: Ca2+ signaling and EC-coupling

    PubMed Central

    Niggli, Ernst; Ullrich, Nina D.; Gutierrez, D.; Kyrychenko, Sergii; Poláková, Eva; Shirokova, Natalia

    2012-01-01

    In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca2+ release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca2+-induced Ca2+ release mechanism and contribute a large fraction of the Ca2+ required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca2+ sensitivity. Presently, research in a number of laboratories is focussed on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS / RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. PMID:22960642

  8. Imaging Single Cardiac Ryanodine Receptor Ca2+ Fluxes in Lipid Bilayers

    PubMed Central

    Peng, S.; Publicover, N. G.; Kargacin, G. J.; Duan, D.; Airey, J. A.; Sutko, John L.

    2004-01-01

    In this and an accompanying report we describe two steps, single-channel imaging and channel immobilization, necessary for using optical imaging to analyze the function of ryanodine receptor (RyR) channels reconstituted in lipid bilayers. An optical bilayer system capable of laser scanning confocal imaging of fluo-3 fluorescence due to Ca2+ flux through single RyR2 channels and simultaneous recording of single channel currents was developed. A voltage command protocol was devised in which the amplitude, time course, shape, and hence the quantity of Ca2+ flux through a single RyR2 channel is controlled solely by the voltage imposed across the bilayer. Using this system, the voltage command protocol, and concentrations of Ca2+ (25–50 mM) that result in saturating RyR2 Ca2+ currents, proportional fluo-3 fluorescence was recorded simultaneously with Ca2+ currents having amplitudes of 0.25–14 pA. Ca2+ sparks, similar to those obtained with conventional microscope-based laser scanning confocal systems, were imaged in mouse ventricular cardiomyocytes using the optical bilayer system. The utility of the optical bilayer for systematic investigation of how cellular factors extrinsic to the RyR2 channel, such as Ca2+ buffers and diffusion, alter fluo-3 fluorescent responses to RyR2 Ca2+ currents, and for addressing other current research questions is discussed. PMID:14695257

  9. A Model of the Putative Pore Region of the Cardiac Ryanodine Receptor Channel

    PubMed Central

    Welch, William; Rheault, Shana; West, Duncan J.; Williams, Alan J.

    2004-01-01

    Using the bacterial K+ channel KcsA as a template, we constructed models of the pore region of the cardiac ryanodine receptor channel (RyR2) monomer and tetramer. Physicochemical characteristics of the RyR2 model monomer were compared with the template, including homology, predicted secondary structure, surface area, hydrophobicity, and electrostatic potential. Values were comparable with those of KcsA. Monomers of the RyR2 model were minimized and assembled into a tetramer that was, in turn, minimized. The assembled tetramer adopts a structure equivalent to that of KcsA with a central pore. Characteristics of the RyR2 model tetramer were compared with the KcsA template, including average empirical energy, strain energy, solvation free energy, solvent accessibility, and hydrophobic, polar, acid, and base moments. Again, values for the model and template were comparable. The pores of KcsA and RyR2 have a common motif with a hydrophobic channel that becomes polar at both entrances. Quantitative comparisons indicate that the assembled structure provides a plausible model for the pore of RyR2. Movement of Ca2+, K+, and tetraethylammonium (TEA+) through the model RyR2 pore were simulated with explicit solvation. These simulations suggest that the model RyR2 pore is permeable to Ca2+ and K+ with rates of translocation greater for K+. In contrast, simulations indicate that tetraethylammonium blocks movement of metal cations. PMID:15454434

  10. Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

    PubMed Central

    Kakizawa, Sho; Yamazawa, Toshiko; Chen, Yili; Ito, Akihiro; Murayama, Takashi; Oyamada, Hideto; Kurebayashi, Nagomi; Sato, Osamu; Watanabe, Masahiko; Mori, Nozomu; Oguchi, Katsuji; Sakurai, Takashi; Takeshima, Hiroshi; Saito, Nobuhito; Iino, Masamitsu

    2012-01-01

    Mobilization of intracellular Ca2+ stores regulates a multitude of cellular functions, but the role of intracellular Ca2+ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca2+ release from intracellular stores of central neurons, and thereby promote prolonged Ca2+ signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca2+ release. NO-induced Ca2+ release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain. PMID:22036948

  11. Voltage-gated Ca2+ entry and ryanodine receptor Ca2+-induced Ca2+ release in preglomerular arterioles.

    PubMed

    Fellner, Susan K; Arendshorst, William J

    2007-05-01

    We have previously shown that in afferent arterioles, angiotensin II (ANG II) involves activation of the inositol trisphosphate receptor (IP(3)R), activation of adenine diphosphoribose (ADPR) cyclase, and amplification of the initial IP(3)R-stimulated release of cytosolic Ca(2+) ([Ca(2+)](i)) from the sarcoplasmic reticulum (SR) (Fellner SK, Arendshorst WJ. Am J Physiol Renal Physiol 288: F785-F791, 2004). The response of the ryanodine receptor (RyR) to local increases in [Ca(2+)](i) is defined as calcium-induced calcium release (CICR). To investigate whether Ca(2+) entry via voltage-gated channels (VGCC) can stimulate CICR, we treated fura 2-loaded, freshly isolated afferent arterioles with KCl (40 mM; high KCl). In control arterioles, peak [Ca(2+)](i) increased by 165 +/- 10 nM. Locking the RyR in the closed position with ryanodine (100 microM) inhibited the [Ca(2+)](i) response by 59% (P < 0.01). 8-Br cADPR, a specific blocker of the ability of cyclic ADPR (cADPR) to sensitize the RyR to Ca(2+), caused a 43% inhibition. We suggest that the lower inhibition by 8-Br cADPR (P = 0.02, ryanodine vs. 8-Br cADPR) represents endogenously active ADPR cyclase. Depletion of SR Ca(2+) stores by inhibiting the SR Ca(2+)-ATPase with cyclopiazonic acid or thapsigargin blocked the [Ca(2+)](i) responses to KCl by 51% (P not significant vs. ryanodine or 8-Br cADPR). These data suggest that about half of the increase in [Ca(2+)](i) induced by high KCl is accomplished by activation of CICR through the ability of entered Ca(2+) to expose the RyR to high local concentrations of Ca(2+) and that endogenous cADPR contributes to the process. PMID:17190906

  12. Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death.

    PubMed

    Marks, A R

    2001-04-01

    Calcium (Ca2+) ions are second messengers in signaling pathways in all types of cells. They regulate muscle contraction, electrical signals which determine the cardiac rhythm and cell growth pathways in the heart. In the past decade cDNA cloning has provided clues as to the molecular structure of the intracellular Ca2+ release channels (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP3R) on the sarcoplasmic and endoplasmic reticulum (SR/ER) and an understanding of how these molecules regulate Ca2+ homeostasis in the heart is beginning to emerge. The intracellular Ca2+ release channels form a distinct class of ion channels distinguished by their structure, size, and function. Both RyRs and IP3Rs have gigantic cytoplasmic domains that serve as scaffolds for modulatory proteins that regulate the channel pore located in the carboxy terminal 10% of the channel sequence. The channels are tetramers comprised of four RyR or IP3R subunits. RyR2 is required for excitation-contraction (EC) coupling in the heart. Using co-sedimentation and co-immunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein mAKAP. We have shown that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (P(o)). In failing human hearts RyR2 is PKA hyperphosphorylated resulting in defective channel function due to increased sensitivity to Ca2+-induced activation. PMID:11273716

  13. Species differences in chlorantraniliprole and flubendiamide insecticide binding sites in the ryanodine receptor.

    PubMed

    Qi, Suzhen; Casida, John E

    2013-11-01

    Anthranilic and phthalic diamides exemplified by chlorantraniliprole (Chlo) or cyantraniliprole (Cyan) and flubendiamide (Flu), respectively, are the newest major chemotype of insecticides with outstanding potency, little or no cross resistance with other classes and low mammalian toxicity. They are activators of the ryanodine (Ry) receptor (RyR)-Ca(2+) channel, based on Ca(2+) flux and electrophysiology investigations. The goal of this study is to define species differences in the degree and mechanisms of diamide selective action by radioligand specific binding studies at the [(3)H]Ry, [(3)H]Chlo and [(3)H]Flu sites. The [(3)H]Ry site is observed in muscle of lobster, rabbit and four insect species (Musca domestica, Apis mellifera, Heliothis virescens and Agrotis ipsilon) whereas the [(3)H]Chlo site is evident in the four insects and the [(3)H]Flu site in only the two lepidoptera (Agrotis and Heliothis). [(3)H]Ry binding is significantly stimulated by Chlo, Cyan and Flu with the insects (except Flu with Musca) but not the lobster and rabbit. [(3)H]Chlo binding is stimulated by Ry and Flu in Musca and Apis but not in the lepidoptera, while Flu and Cyan are inhibitory. [(3)H]Flu binding is strongly inhibited by Chlo and Cyan in Agrotis and Heliothis. [(3)H]Chlo and [(3)H]Flu binding are not dependent on added Ca(2+) or ATP in Heliothis and Agrotis whereas the other radioligand-receptor combinations are usually enhanced by Ca(2+) and ATP. More generally, there are species differences in the Ry, Chlo and Flu binding sites of the RyR that may confer selective toxicity and determine target site cross resistance mechanisms. PMID:24267693

  14. S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction

    PubMed Central

    Ritterhoff, Julia; Völkers, Mirko; Seitz, Andreas; Spaich, Kristin; Gao, Erhe; Peppel, Karsten; Pleger, Sven T; Zimmermann, Wolfram H; Friedrich, Oliver; Fink, Rainer H A; Koch, Walter J; Katus, Hugo A; Most, Patrick

    2015-01-01

    Restoring expression levels of the EF-hand calcium (Ca2+) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca2+ handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca2+- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca2+ leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca2+ leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca2+ leak in HF, combining antiarrhythmic potency with chronic inotropic actions. PMID:26005840

  15. Structural and functional conservation of key domains in InsP[subscript 3] and ryanodine receptors

    SciTech Connect

    Seo, Min-Duk; Velamakanni, Saroj; Ishiyama, Noboru; Stathopulos, Peter B.; Rossi, Ana M.; Khan, Samir A.; Dale, Philippa; Li, Congmin; Ames, James B.; Ikura, Mitsuhiko; Taylor, Colin W.

    2012-07-11

    Inositol-1,4,5-trisphosphate receptors (InsP{sub 3}Rs) and ryanodine receptors (RyRs) are tetrameric intracellular Ca{sup 2+} channels. In each of these receptor families, the pore, which is formed by carboxy-terminal transmembrane domains, is regulated by signals that are detected by large cytosolic structures. InsP{sub 3}R gating is initiated by InsP{sub 3} binding to the InsP{sub 3}-binding core (IBC, residues 224-604 of InsP{sub 3}R1) and it requires the suppressor domain (SD, residues 1-223 of InsP{sub 3}R1). Here we present structures of the amino-terminal region (NT, residues 1-604) of rat InsP{sub 3}R1 with (3.6 {angstrom}) and without (3.0 {angstrom}) InsP{sub 3} bound. The arrangement of the three NT domains, SD, IBC-{beta} and IBC-{alpha}, identifies two discrete interfaces ({alpha} and {beta}) between the IBC and SD. Similar interfaces occur between equivalent domains (A, B and C) in RyR1 (ref. 9). The orientations of the three domains when docked into a tetrameric structure of InsP{sub 3}R and of the ABC domains docked into RyR are remarkably similar. The importance of the {alpha}-interface for activation of InsP{sub 3}R and RyR is confirmed by mutagenesis and, for RyR, by disease-causing mutations. Binding of InsP{sub 3} causes partial closure of the clam-like IBC, disrupting the {beta}-interface and pulling the SD towards the IBC. This reorients an exposed SD loop ('hotspot' (HS) loop) that is essential for InsP{sub 3}R activation. The loop is conserved in RyR and includes mutations that are associated with malignant hyperthermia and central core disease. The HS loop interacts with an adjacent NT, suggesting that activation re-arranges inter-subunit interactions. The A domain of RyR functionally replaced the SD in full-length InsP{sub 3}R, and an InsP{sub 3}R in which its C-terminal transmembrane region was replaced by that from RyR1 was gated by InsP{sub 3} and blocked by ryanodine. Activation mechanisms are conserved between InsP{sub 3}R and Ry

  16. Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.

    PubMed Central

    Ahern, G P; Junankar, P R; Dulhunty, A F

    1997-01-01

    FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs. Images FIGURE 1 PMID:8994600

  17. Ryanodine Receptor Activation Induces Long-Term Plasticity of Spine Calcium Dynamics.

    PubMed

    Johenning, Friedrich W; Theis, Anne-Kathrin; Pannasch, Ulrike; Rückl, Martin; Rüdiger, Sten; Schmitz, Dietmar

    2015-06-01

    A key feature of signalling in dendritic spines is the synapse-specific transduction of short electrical signals into biochemical responses. Ca2+ is a major upstream effector in this transduction cascade, serving both as a depolarising electrical charge carrier at the membrane and an intracellular second messenger. Upon action potential firing, the majority of spines are subject to global back-propagating action potential (bAP) Ca2+ transients. These transients translate neuronal suprathreshold activation into intracellular biochemical events. Using a combination of electrophysiology, two-photon Ca2+ imaging, and modelling, we demonstrate that bAPs are electrochemically coupled to Ca2+ release from intracellular stores via ryanodine receptors (RyRs). We describe a new function mediated by spine RyRs: the activity-dependent long-term enhancement of the bAP-Ca2+ transient. Spines regulate bAP Ca2+ influx independent of each other, as bAP-Ca2+ transient enhancement is compartmentalized and independent of the dendritic Ca2+ transient. Furthermore, this functional state change depends exclusively on bAPs travelling antidromically into dendrites and spines. Induction, but not expression, of bAP-Ca2+ transient enhancement is a spine-specific function of the RyR. We demonstrate that RyRs can form specific Ca2+ signalling nanodomains within single spines. Functionally, RyR mediated Ca2+ release in these nanodomains induces a new form of Ca2+ transient plasticity that constitutes a spine specific storage mechanism of neuronal suprathreshold activity patterns. PMID:26098891

  18. Reversible redox modifications of ryanodine receptor ameliorate ventricular arrhythmias in the ischemic-reperfused heart.

    PubMed

    Becerra, Romina; Román, Bárbara; Di Carlo, Mariano N; Mariangelo, Juan Ignacio; Salas, Margarita; Sanchez, Gina; Donoso, Paulina; Schinella, Guillermo R; Vittone, Leticia; Wehrens, Xander H; Mundiña-Weilenmann, Cecilia; Said, Matilde

    2016-09-01

    Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca(2+) calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide synthase. Left ventricular contractile parameters and monophasic action potentials were recorded. Oxidation and phosphorylation of CaMKII and RyR2 were assessed. Increased oxidation of CaMKII during reperfusion had no consequences on the level of RyR2 phosphorylation. Avoiding the reperfusion-induced thiol oxidation of RyR2 with MPG produced a reduction in the number of arrhythmias and did not modify the contractile recovery. Conversely, selective prevention of S-nitrosylation and S-glutathionylation of RyR2 was associated with higher numbers of arrhythmias and impaired contractility. In S2814A mice, treatment with MPG further reduced the incidence of arrhythmias. Taken together, the results suggest that redox modification of RyR2 synergistically with CaMKII phosphorylation modulates reperfusion arrhythmias. PMID:27422983

  19. Chiral dicarboxamide scaffolds containing a sulfiliminyl moiety as potential ryanodine receptor activators.

    PubMed

    Zhou, Sha; Jia, Zhehui; Xiong, Lixia; Yan, Tao; Yang, Na; Wu, Guiping; Song, Haibin; Li, Zhengming

    2014-07-01

    To search for new environmentally benign insecticides with high activity, low toxicity, and low residue, novel chiral configurations introduced into dicarboxamide scaffolds containing N-cyano sulfiliminyl moieties were first studied. Four series of phthalamides with sulfur-containing side chains were designed, synthesized, and evaluated against oriental armyworm (Pseudaletia separata Walker) and diamondback moth (Plutella xylostella (L.)) for their insecticidal activities. All structures were characterized by (1)H NMR, (13)C NMR, and HRMS (or elemental analysis), and their configurations were confirmed by optical polarimetry. The biological assessment indicated that some title compounds exhibited significant insecticidal activities. For oriental armyworm, these stereoisomers exerted different impacts on biological activity following the sequence (Sc, Ss) ≥ (Sc, Rs) ≫ (Rc, Ss) > (Rc, Rs), and carbon chirality influenced the activities more strongly than sulfur. Compounds Ia and IIa reached as high an activity as commercial flubendiamide, with LC50 values of 0.0504 and 0.0699 mg L(-1), respectively, lower than that of flubendiamide (0.1230 mg L(-1)). For diamondback moth, the sequence of activity was (Sc, Ss) > (Sc, Rs), and the sulfur chirality influenced the activities more greatly than carbon. Compound IIe exhibited even higher activity than flubendiamide, whereas Ie and Ic,d reached the activity of the latter. The results indicated that the improvement of insecticidal activity probably required a coordination of both carbon and sulfur chirality. Comparative molecular field analysis calculation indicated that stereoisomers with Sc configurations containing strong electron-withdrawing groups such as as CN are important in maintaining the high activity. The chiral scaffolds containing the N-cyano sulfiliminyl moiety are also essential for high larvicidal activity. Some title compounds could be considered as potential candidates for ryanodine receptor activators

  20. Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion.

    PubMed

    Pedrozo, Zully; Sánchez, Gina; Torrealba, Natalia; Valenzuela, Rodrigo; Fernández, Carolina; Hidalgo, Cecilia; Lavandero, Sergio; Donoso, Paulina

    2010-03-01

    Type-2 ryanodine receptors (RyR2)--the calcium release channels of cardiac sarcoplasmic reticulum--have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems--calpains, the proteasome and autophagy--on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia. PMID:20026269

  1. Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

    PubMed Central

    2011-01-01

    Background Calcium signals ([Ca2+]i) direct many aspects of embryo development but their regulation is not well characterised. Ryanodine receptors (RyRs) are a family of intracellular Ca2+ release channels that control the flux of Ca2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation. Findings The sequences of the five zebrafish ryr genes were confirmed, their evolutionary relationship established and the primary sequences compared to other vertebrates, including humans. RyRs are differentially expressed in slow (ryr1a), fast (ryr3) and both types (ryr1b) of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b) which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation. Conclusions Our work reveals that zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types. PMID:22168922

  2. Three-Dimensional Distribution of Ryanodine Receptor Clusters in Cardiac Myocytes

    PubMed Central

    Chen-Izu, Ye; McCulle, Stacey L.; Ward, Chris W.; Soeller, Christian; Allen, Bryan M.; Rabang, Cal; Cannell, Mark B.; Balke, C. William; Izu, Leighton T.

    2006-01-01

    The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 μm in ventricular myocytes and estimated 0.97 μm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 μm in ventricular and 1.69 μm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that ∼100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed. PMID:16603500

  3. Blockage of the Ryanodine Receptor via Azumolene Does Not Prevent Mechanical Ventilation-Induced Diaphragm Atrophy

    PubMed Central

    Talbert, Erin E.; Smuder, Ashley J.; Kwon, Oh Sung; Sollanek, Kurt J.; Wiggs, Michael P.; Powers, Scott K.

    2016-01-01

    Mechanical ventilation (MV) is a life-saving intervention for patients in respiratory failure. However, prolonged MV causes the rapid development of diaphragm muscle atrophy, and diaphragmatic weakness may contribute to difficult weaning from MV. Therefore, developing a therapeutic countermeasure to protect against MV-induced diaphragmatic atrophy is important. MV-induced diaphragm atrophy is due, at least in part, to increased production of reactive oxygen species (ROS) from diaphragm mitochondria and the activation of key muscle proteases (i.e., calpain and caspase-3). In this regard, leakage of calcium through the ryanodine receptor (RyR1) in diaphragm muscle fibers during MV could result in increased mitochondrial ROS emission, protease activation, and diaphragm atrophy. Therefore, these experiments tested the hypothesis that a pharmacological blockade of the RyR1 in diaphragm fibers with azumolene (AZ) would prevent MV-induced increases in mitochondrial ROS production, protease activation, and diaphragmatic atrophy. Adult female Sprague-Dawley rats underwent 12 hours of full-support MV while receiving either AZ or vehicle. At the end of the experiment, mitochondrial ROS emission, protease activation, and fiber cross-sectional area were determined in diaphragm muscle fibers. Decreases in muscle force production following MV indicate that the diaphragm took up a sufficient quantity of AZ to block calcium release through the RyR1. However, our findings reveal that AZ treatment did not prevent the MV-induced increase in mitochondrial ROS emission or protease activation in the diaphragm. Importantly, AZ treatment did not prevent MV-induced diaphragm fiber atrophy. Thus, pharmacological inhibition of the RyR1 in diaphragm muscle fibers is not sufficient to prevent MV-induced diaphragm atrophy. PMID:26849371

  4. Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum.

    PubMed

    Seo, In-Ra; Moh, Sang Hyun; Lee, Eun Hui; Meissner, Gerhard; Kim, Do Han

    2006-10-15

    DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1. PMID:16817780

  5. Chronic diabetes increases advanced glycation end products on cardiac ryanodine receptors/calcium-release channels.

    PubMed

    Bidasee, Keshore R; Nallani, Karuna; Yu, Yongqi; Cocklin, Ross R; Zhang, Yinong; Wang, Mu; Dincer, U Deniz; Besch, Henry R

    2003-07-01

    Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H(+)) >or=500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H(+) peaks corresponding to theoretical RyR2 peptides with single N(epsilon)-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction. PMID:12829653

  6. Ryanodine Receptor Activation Induces Long-Term Plasticity of Spine Calcium Dynamics

    PubMed Central

    Pannasch, Ulrike; Rückl, Martin; Rüdiger, Sten; Schmitz, Dietmar

    2015-01-01

    A key feature of signalling in dendritic spines is the synapse-specific transduction of short electrical signals into biochemical responses. Ca2+ is a major upstream effector in this transduction cascade, serving both as a depolarising electrical charge carrier at the membrane and an intracellular second messenger. Upon action potential firing, the majority of spines are subject to global back-propagating action potential (bAP) Ca2+ transients. These transients translate neuronal suprathreshold activation into intracellular biochemical events. Using a combination of electrophysiology, two-photon Ca2+ imaging, and modelling, we demonstrate that bAPs are electrochemically coupled to Ca2+ release from intracellular stores via ryanodine receptors (RyRs). We describe a new function mediated by spine RyRs: the activity-dependent long-term enhancement of the bAP-Ca2+ transient. Spines regulate bAP Ca2+ influx independent of each other, as bAP-Ca2+ transient enhancement is compartmentalized and independent of the dendritic Ca2+ transient. Furthermore, this functional state change depends exclusively on bAPs travelling antidromically into dendrites and spines. Induction, but not expression, of bAP-Ca2+ transient enhancement is a spine-specific function of the RyR. We demonstrate that RyRs can form specific Ca2+ signalling nanodomains within single spines. Functionally, RyR mediated Ca2+ release in these nanodomains induces a new form of Ca2+ transient plasticity that constitutes a spine specific storage mechanism of neuronal suprathreshold activity patterns. PMID:26098891

  7. Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

    PubMed Central

    Bauerová-Hlinková, Vladena; Hostinová, Eva; Gašperík, Juraj; Beck, Konrad; Borko, Ľubomír; Lai, F. Anthony; Zahradníková, Alexandra; Ševčík, Jozef

    2010-01-01

    We report the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR21–606·His6, RyR2391–606·His6, RyR2409–606·His6, Trx·RyR2384–606·His6, Trx·RyR2391-606·His6 and Trx·RyR2409–606·His6. The folding of RyR21–606·His6 was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR21–606·His6, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His6·Tag antibody indicated that RyR21–606·His6 is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively. PMID:20045464

  8. Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

    PubMed Central

    Macquaide, Niall; Tuan, Hoang-Trong Minh; Hotta, Jun-ichi; Sempels, Wouter; Lenaerts, Ilse; Holemans, Patricia; Hofkens, Johan; Jafri, M. Saleet; Willems, Rik; Sipido, Karin R.

    2015-01-01

    Aims In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and results Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release. PMID:26490742

  9. Cardiomyocytes hypertrophic status after myocardial infarction determines distinct types of arrhythmia: role of the ryanodine receptor.

    PubMed

    Fauconnier, Jérémy; Pasquié, Jean-Luc; Bideaux, Patrice; Lacampagne, Alain; Richard, Sylvain

    2010-09-01

    The mechanisms responsible for sudden cardiac death in heart failure (HF) are unclear. We investigated early and delayed afterdepolarizations (EADs, DADs) in HF. Cardiomyocytes were enzymatically isolated from the right ventricle (RV) and the septum of rats 8 weeks after myocardial infarction (MI) and sham-operated animals. Membrane capacitance, action potentials (AP) and ionic currents were measured by whole-cell patch-clamp. The [Ca(2+)](i) transients and Ca(2+) sparks were recorded with Fluo-4 during fluorescence measurements. Arrhythmia was triggered in 40% of MI cells (not in sham) using trains of 5 stimulations at 2.0 Hz. EADs and DADs occurred in distinct cell populations both in the RV and the septum. EADs occurred in normal-sized PMI cells (<230 pF), whereas DADs occurred in hypertrophic PMI cells (>230 pF). All cells exhibited prolonged APs due to reduced I(to) current. However, additional modifications in Ca(2+)-dependent ionic currents occurred in hypertrophic cells: a decrease in the inward rectifier K(+) current I(K1), and a slowing of L-type Ca(2+) current inactivation which was responsible for the lack of adaptation of APs to abrupt changes in the pacing rate. The occurrence of spontaneous Ca(2+) sparks, reflecting ryanodine receptor (RyR2) diastolic activity, increased with hypertrophy. The [Ca(2+)](i) transient amplitude, sarcoplasmic reticulum (SR) Ca(2+) load and Ca(2+) sparks amplitude were all inversely correlated with cell size. We conclude that the trophic status of cardiomyocytes determines the type of cellular arrhythmia in MI rats, based on differential electrophysiological remodeling which may reflect early-mild and late-severe or differential modifications in the RyR2 function. PMID:20109482

  10. The role of ryanodine receptor type 3 in a mouse model of Alzheimer disease.

    PubMed

    Liu, Jie; Supnet, Charlene; Sun, Suya; Zhang, Hua; Good, Levi; Popugaeva, Elena; Bezprozvanny, Ilya

    2014-01-01

    Dysregulated endoplasmic reticulum (ER) calcium (Ca(2+)) signaling is reported to play an important role in Alzheimer disease (AD) pathogenesis. The role of ER Ca(2+) release channels, the ryanodine receptors (RyanRs), has been extensivelys tudied in AD models and RyanR expression and activity are upregulated in the brains of various familial AD (FAD) models.The objective of this study was to utilize a genetic approach to evaluate the importance of RyanR type 3 (RyanR3) in the context of AD pathology.The expression of RyanR3 was also elevated in hippocampus of APPPS1 mice (Thy1-APPKM670/671NL, Thy1-PS1L166P).In young (≤ 3 mo) APPPS1 mice, the deletion of RyanR3 increased hippocampal neuronal network excitability and accelerated AD pathology, leading to mushroom spine loss and increased amyloid accumulation. In contrast, deletion of RyanR3 in older APPPS1 mice (≥ 6 mo) rescued network excitability and mushroom spine loss, reduced amyloid plaque load and reduced spontaneous seizure occurrence.Our data suggests a dual role for RyanR3 in AD pathology. In young AD neurons, RyanR3 protects AD neurons from synaptic and network dysfunction. In older AD neurons, increased RyanR3 activity contributes to pathology. These results imply that blockade of RyanR3 may be beneficial for those in the later stages of the disease, but RyanR activators may be beneficial when used prior to disease onset or in its initial stages. Caffeine is an activator of RyanRs and our results may help to explain a complex epidemiological connection between coffee consumption in mid-life and risk of AD development in old age. PMID:24476841

  11. Peptide fragments of the dihydropyridine receptor can modulate cardiac ryanodine receptor channel activity and sarcoplasmic reticulum Ca2+ release.

    PubMed Central

    Dulhunty, Angela F; Curtis, Suzanne M; Cengia, Louise; Sakowska, Magdalena; Casarotto, Marco G

    2004-01-01

    We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers. PMID:14678014

  12. A novel mutation (Arg169Gln) of the cardiac ryanodine receptor gene causing exercise-induced bidirectional ventricular tachycardia.

    PubMed

    Hsueh, Chia-Hsiang; Weng, Yi-Chun; Chen, Chao-Yu; Lin, Tin-Kwang; Lin, Yen-Hung; Lai, Ling-Ping; Lin, Jiunn-Lee

    2006-04-01

    An 18-year-old woman presented with exercise induced sudden collapse. Series of cardiac work up revealed no structural cardiac abnormalities. Bidirectional ventricular tachycardia occurred during a treadmill exercise test. Under the impression of catecholaminergic polymorphic ventricular tachycardia, we screened the cardiac ryanodine receptor gene for mutation. We identified a novel heterozygous mutation at the 169th amino acid (Arg169Gln). This amino acid is highly conserved among many species and this mutation was not present in 50 normal control subjects. This patient was treated with a beta-block with good response. PMID:16517285

  13. One Dimensional Finite Element Method Approach to Study Effect of Ryanodine Receptor and Serca Pump on Calcium Distribution in Oocytes

    NASA Astrophysics Data System (ADS)

    Naik, Parvaiz Ahmad; Pardasani, Kamal Raj

    2013-11-01

    Oocyte is a female gametocyte or germ cell involved in reproduction. Calcium ions (Ca2+) impact nearly all aspects of cellular life as they play an important role in a variety of cellular functions. Calcium ions contributes to egg activation upon fertilization. Since it is the internal stores which provide most of the calcium signal, much attention has been focused on the intracellular channels. There are mainly two types of calcium channels which release calcium from the internal stores to the cytoplasm in many cell types. These channels are IP3-Receptor and Ryanodine Receptor (RyR). Further it is essential to maintain low cytosolic calcium concentration, the cell engages the Serco/Endoplasmic reticulum Ca2+ ATPases (SERCA) present on the ER or SR membrane for the re-uptake of cytosolic calcium at the expense of ATP hydrolysis. In view of above an attempt has been made to study the effect of the Ryanodine receptor (RyR) and the SERCA pump on the calcium distribution in oocytes. The main aim of this paper is to study the calcium concentration in absence and presence of these parameters. The FEM is used to solve the proposed Mathematical model under appreciate initial and boundary conditions. The program has been developed in MATLAB 7.10 for the entire problem to get numerical results.

  14. Apolipoprotein E isoform-specific effects on lipoprotein receptor processing

    PubMed Central

    Bachmeier, Corbin; Shackleton, Ben; Ojo, Joseph; Paris, Daniel; Mullan, Michael; Crawford, Fiona

    2014-01-01

    Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood-brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained elevated. Likewise, intracranial administration of Aβ to apoE targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4>apoE3>apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain. PMID:25015123

  15. NAADP-mediated Ca2+ signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist.

    PubMed

    Dammermann, Werner; Zhang, Bo; Nebel, Merle; Cordiglieri, Chiara; Odoardi, Francesca; Kirchberger, Tanja; Kawakami, Naoto; Dowden, James; Schmid, Frederike; Dornmair, Klaus; Hohenegger, Martin; Flügel, Alexander; Guse, Andreas H; Potter, Barry V L

    2009-06-30

    The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca(2+) signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca(2+) mobilization by d-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca(2+) entry. BZ194 specifically and effectively blocked NAADP-stimulated [(3)H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca(2+) mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca(2+) mobilization, such as nuclear translocation of "nuclear factor of activated T cells" (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4(+) effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases. PMID:19541638

  16. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells.

    PubMed

    Stern, Michael D; Maltseva, Larissa A; Juhaszova, Magdalena; Sollott, Steven J; Lakatta, Edward G; Maltsev, Victor A

    2014-05-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This "calcium clock" takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic-mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in beating

  17. Unbalanced upregulation of ryanodine receptor 2 plays a particular role in early development of daunorubicin cardiomyopathy

    PubMed Central

    Kucerova, Dana; Doka, Gabriel; Kruzliak, Peter; Turcekova, Katarina; Kmecova, Jana; Brnoliakova, Zuzana; Kyselovic, Jan; Kirchhefer, Uwe; Müller, Frank U; Krenek, Peter; Boknik, Peter; Klimas, Jan

    2015-01-01

    Calcium release channel on the sarcoplasmic reticulum of cardiomyocytes (ryanodine receptor type 2, RyR2) plays a critical role in the regulation of calcium and was identified as a crucial factor for development of chronic anthracycline cardiomyopathy. Its early stages are less well described although these determine the later development. Hence, we tested the effect of repeated, short-term anthracycline (daunorubicin) administration on cardiac performance, cardiomyocyte function and accompanied changes in calcium regulating proteins expression. Ten-twelve weeks old male Wistar rats were administered with 6 doses of daunorubicin (DAU, 3 mg/kg, i.p., every 48 h), controls (CON) received vehicle. Left ventricular function (left ventricular pressure, LVP; rate of pressure development, +dP/dt and decline, -dP/dt) was measured using left ventricular catheterization under tribromethanol anaesthesia (15 ml/kg b.w.). Cell shortening was measured in enzymatically isolated cardiomyocytes. The expressions of RyR2 and associated intracellular calcium regulating proteins, cytoskeletal proteins (alpha-actinin, alpha-tubul in) as well as oxidative stress regulating enzymes (gp91phox, MnSOD) were detected in ventricular tissue samples using immunoblotting. mRNA expressions of cardiac damage markers (Nppa and Nppb, atrial and brain natriuretic peptides; Myh6, Myh7 and Myh7b, myosin heavy chain alpha and beta) were detected using RT-PCR. Thiobarbituric acid reactive substances concentration was measured to estimate oxidative stress. DAU rats exhibited significantly depressed left ventricular features (LVP by 14%, +dP/dt by 36% and -dP/dt by 30%; for all P<0.05), in line with concomitant increase in Nppa and Nppb gene expressions (3.23- and 2.18-fold, for both P<0.05), and a 4.34-fold increase in Myh7 (P<0.05). Controversially, we observed increased cell shortening of isolated cardiac cells by 31% (p<0.05). DAU administration was associated with a twofold upregulation of RyR2 (P<0

  18. Hierarchical clustering of ryanodine receptors enables emergence of a calcium clock in sinoatrial node cells

    PubMed Central

    Maltseva, Larissa A.; Juhaszova, Magdalena; Sollott, Steven J.; Lakatta, Edward G.; Maltsev, Victor A.

    2014-01-01

    The sinoatrial node, whose cells (sinoatrial node cells [SANCs]) generate rhythmic action potentials, is the primary pacemaker of the heart. During diastole, calcium released from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) interacts with membrane currents to control the rate of the heartbeat. This “calcium clock” takes the form of stochastic, partially periodic, localized calcium release (LCR) events that propagate, wave-like, for limited distances. The detailed mechanisms controlling the calcium clock are not understood. We constructed a computational model of SANCs, including three-dimensional diffusion and buffering of calcium in the cytosol and SR; explicit, stochastic gating of individual RyRs and L-type calcium channels; and a full complement of voltage- and calcium-dependent membrane currents. We did not include an anatomical submembrane space or inactivation of RyRs, the two heuristic components that have been used in prior models but are not observed experimentally. When RyRs were distributed in discrete clusters separated by >1 µm, only isolated sparks were produced in this model and LCR events did not form. However, immunofluorescent staining of SANCs for RyR revealed the presence of bridging RyR groups between large clusters, forming an irregular network. Incorporation of this architecture into the model led to the generation of propagating LCR events. Partial periodicity emerged from the interaction of LCR events, as observed experimentally. This calcium clock becomes entrained with membrane currents to accelerate the beating rate, which therefore was controlled by the activity of the SERCA pump, RyR sensitivity, and L-type current amplitude, all of which are targets of β-adrenergic–mediated phosphorylation. Unexpectedly, simulations revealed the existence of a pathological mode at high RyR sensitivity to calcium, in which the calcium clock loses synchronization with the membrane, resulting in a paradoxical decrease in

  19. Regulation of Ca2+ Signaling in Rat Bile Duct Epithelia by Inositol 1,4,5-Trisphosphate Receptor Isoforms

    PubMed Central

    Hirata, Keiji; Dufour, Jean-François; Shibao, Kazunori; Knickelbein, Roy; O'Neill, Allison F.; Bode, Hans-Peter; Cassio, Doris; St-Pierre, Marie V.; LaRusso, Nicholas F.; Leite, M. Fatima; Nathanson, Michael H.

    2010-01-01

    Cytosolic Ca2+ (Cai2+) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca2+ waves, Ca2+ oscillations, and other subcellular Ca2+ signaling patterns, but little is known about the subcellular organization of Ca2+ signaling in cholangiocytes. Therefore, we examined Ca2+ signaling and the subcellular distribution of Ca2+ release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca2+ signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP3R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP3R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP3R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca2+ via InsP3 in cholangiocytes, induced Ca2+ oscillations in both cholangiocytes and NRC cells. Acetylcholine (ACh) induced apical-to-basal Ca2+ waves. In conclusion, Ca2+ signaling in cholangiocytes occurs as polarized Ca2+ waves that begin in the region of the type III InsP3R. Differential subcellular localization of InsP3R isoforms may be an important molecular mechanism for the formation of Ca2+ waves and oscillations in cholangiocytes. Because Cai2+ is in part responsible for regulating ductular secretion, these findings also may have implications for the molecular basis of cholestatic disorders. PMID:12143036

  20. Discovery of cyantraniliprole, a potent and selective anthranilic diamide ryanodine receptor activator with cross-spectrum insecticidal activity.

    PubMed

    Selby, Thomas P; Lahm, George P; Stevenson, Thomas M; Hughes, Kenneth A; Cordova, Daniel; Annan, I Billy; Barry, James D; Benner, Eric A; Currie, Martin J; Pahutski, Thomas F

    2013-12-01

    Anthranilic diamides are an exceptionally active class of insect control chemistry that selectively activates insect ryanodine receptors causing mortality from uncontrolled release of calcium ion stores in muscle cells. Work in this area led to the successful commercialization of chlorantraniliprole for control of Lepidoptera and other insect pests at very low application rates. In search of lower logP analogs with improved plant systemic properties, exploration of cyano-substituted anthranilic diamides culminated in the discovery of a second product candidate, cyantraniliprole, having excellent activity against a wide range of pests from multiple insect orders. Here we report on the chemistry, biology and structure-activity trends for a series of cyanoanthranilic diamides from which cyantraniliprole was selected for commercial development. PMID:24135728

  1. RNAi suppression of the ryanodine receptor gene results in decreased susceptibility to chlorantraniliprole in Colorado potato beetle Leptinotarsa decemlineata.

    PubMed

    Wan, Pin-Jun; Guo, Wei-Yan; Yang, Yao; Lü, Feng-Gong; Lu, Wei-Ping; Li, Guo-Qing

    2014-04-01

    Leptinotarsadecemlineata is the most important pest in potato and causes serious yield loss each year. Chlorantraniliprole acts on insect ryanodine receptors (RyRs) and is among the most active compounds against L. decemlineata. Here we cloned and characterized a 15,792-bp full-length LdRyR cDNA that encoded a 5128-amino acid protein. LdRyR shares 85-92% amino acid similarities with other insect RyR homologues, and 59-61% similarities with those from Caenorhabditis elegans and Homo sapiens. All hallmarks of the RyR proteins are conserved in LdRyR. LdRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR domain and a RIH-associated domain in the N-terminus, and it possesses two consensus calcium ion-binding EF-hand motifs and six predicted transmembrane helices in the C-terminus. Temporal, spatial and tissue-specific expression patterns of LdRyR were evaluated. LdRyR expression level was increased constantly from egg to wandering stages, dropped in pupal stage and was increased again in the adult stage. It was widely expressed in the head, thorax and abdomen of day 3 fourth-instar larvae. Moreover, it was ubiquitously expressed in all inspected tissues including epidermis, foregut, midgut, ileum, rectum, fat body, ventral ganglia and Malpighian tubules in day 3 fourth-instar larvae. Dietary introduction of double-stranded RNA of LdRyR significantly reduced the mRNA levels of the target gene in the larvae and adults, respectively, and significantly decreased chlorantraniliprole-induced mortalities. Thus, our results suggested that LdRyR encoded a functional ryanodine receptor in L. decemlineata. PMID:24607641

  2. Rapid cooling-induced contractures in rat skinned skeletal muscle fibres originate from sarcoplasmic reticulum Ca2+ release through ryanodine and inositol trisphosphate receptors.

    PubMed

    Talon, S; Huchet-Cadiou, C; Léoty, C

    2000-11-01

    Previous reports have shown that cooling striated muscles induces contractile responses that are related to Ca2+ release from the sarcoplasmic reticulum. However, the effect of cooling has generally been studied in the presence of pharmacological agents that potentiate rapid cooling-induced contractures. The present study shows that in saponin-skinned rat skeletal muscle preparations, a drop in temperature from 22 degrees C to 2 degrees C per se induces a contracture which relaxes on return to 22 degrees C. In fast-twitch fibres, rapid cooling-induced contractures are fully blocked by ryanodine, an inhibitor of ryanodine receptors. By contrast, in slow-twitch fibres, ryanodine partially inhibits the rapid cooling-induced contractile response, leaving a residual tension that dissipates after application of inositol 1,4,5-trisphosphate (InsP3). At low concentrations, heparin, an inhibitor of InsP3 receptors, decreases rapid cooling-induced contractures in both types of muscle. The present results suggest that in skeletal muscle, rapid cooling-induced contractures are due to both ryanodine-sensitive and InsP3-sensitive Ca2+ release from the sarcoplasmic reticulum. PMID:11205048

  3. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts

    PubMed Central

    O-Uchi, Jin; Jhun, Bong Sook; Hurst, Stephen; Bisetto, Sara; Gross, Polina; Chen, Ming; Kettlewell, Sarah; Park, Jongsun; Oyamada, Hideto; Smith, Godfrey L.; Murayama, Takashi

    2013-01-01

    Ca+ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca2+ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca2+ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca2+ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca2+ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca2+ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca2+-induced ATP production in cardiac H9c2 myoblasts. PMID:24124188

  4. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  5. Specific roles of GABAB(1) receptor isoforms in cognition

    PubMed Central

    Jacobson, Laura H.; Kelly, Peter H.; Bettler, Bernhard; Kaupmann, Klemens; Cryan, John F.

    2010-01-01

    The GABAB receptor is a heterodimer of GABAB(1) and GABAB(2) subunits. There are two isoforms of the GABAB(1) subunit: GABAB(1a) and GABAB(1b). Recent studies with mutant mice suggest a differential role for the two GABAB(1) isoforms in behavioural processes. As pharmacological and genetic studies have implicated GABAB receptors in cognition we investigated the behaviour of GABAB(1a) −/− and GABAB(1b) −/− mice in different types of cognitive paradigms. GABAB(1a) −/− and GABAB(1b) −/− mice were both impaired relative to wildtype controls in a continuous spontaneous alternation behaviour test of working spatial memory. In contrast to the reported phenotype of GABAB(1) −/− mice, however, neither GABAB(1a) −/− nor GABAB(1b) −/− mice were deficient in a passive avoidance task. On the other hand, GABAB(1a) −/− mice were impaired in familiar and novel object recognition. We conclude that GABAB(1) isoforms contribute differentially to GABAB receptor-mediated cognitive processes. PMID:17498817

  6. Pre-Slaughter Stress Affects Ryanodine Receptor Protein Gene Expression and the Water-Holding Capacity in Fillets of the Nile Tilapia

    PubMed Central

    Lara, Jorge A. F.; Gasparino, Eliane; Del Vesco, Ana P.; Goes, Marcio D.; Alexandre Filho, Luiz

    2015-01-01

    Current study evaluated the effect of pre-slaughter stress on serum cortisol levels, pH, colorimetry, water-holding capacity (WHC) and gene expression of ryanodine receptors (RyR1 and RyR3) in the Nile tilapia. A 3x4 factorial scheme experiment was conducted comprising three densities (100, 200, 400 kg/m³) with four transportation times (60, 120, 180, and 240 minutes).Transportation times alone reduced cortisol levels up to 180 minutes, followed by increased WHC and mRNA expression, RyR1 and RyR3 (200 kg/m³ density). No effect of density x transportation time interacted on the evaluated parameters. Results provided the first evidence that pre-slaughter stress affected ryanodine gene expression receptors and, consequently, the water-holding capacity in tilapia fillets. PMID:26053858

  7. Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

    PubMed Central

    Chung, Kyung Min; Jeong, Eun-Ji; Park, Hyunhee; An, Hyun-Kyu; Yu, Seong-Woon

    2016-01-01

    Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (IP3Rs), the main Ca2+ release channels located in endoplasmic reticulum (ER) membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN) cells utilizing an insulin withdrawal model of autophagic cell death (ACD). Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs—especially RyR3—were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished ACD of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology. PMID:27199668

  8. Electron-conformational transformations govern the temperature dependence of the cardiac ryanodine receptor gating

    NASA Astrophysics Data System (ADS)

    Moskvin, A. S.; Iaparov, B. I.; Ryvkin, A. M.; Solovyova, O. E.; Markhasin, V. S.

    2015-07-01

    Temperature influences many aspects of cardiac excitation-contraction coupling, in particular, hypothermia increases the open probability ( P open) of cardiac sarcoplasmic reticulum (SR) Ca2+-release channels (ryanodine-sensitive RyR channels) rising the SR Ca2+ load in mammalian myocytes. However, to the best of our knowledge, no theoretical models are available for that effect. Traditional Markov chain models do not provide a reasonable molecular mechanistic insight on the origin of the temperature effects. Here in the paper we address a simple physically clear electron-conformational model to describe the RyR gating and argue that a synergetic effect of external thermal fluctuation forces (Gaussian-Markovian noise) and internal friction via the temperature stimulation/suppression of the open-close RyR tunneling probability can be considered as a main contributor to temperature effects on the RyR gating. Results of the computer modeling allowed us to successfully reproduce all the temperature effects observed for an isolated RyR gating in vitro under reducing the temperature: increase in P open and mean open time without any significant effect on mean closed

  9. Knockout of the BK β4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability.

    PubMed

    Wang, Bin; Bugay, Vladislav; Ling, Ling; Chuang, Hui-Hsui; Jaffe, David B; Brenner, Robert

    2016-08-01

    BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK β4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of β4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the β4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons. PMID:27146987

  10. Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats.

    PubMed

    Morel, Jean-Luc; Dabertrand, Fabrice; Porte, Yves; Prevot, Anne; Macrez, Nathalie

    2014-08-01

    Microgravity induces a redistribution of blood volume. Consequently, astronauts' body pressure is modified so that the upright blood pressure gradient is abolished, thereby inducing a modification in cerebral blood pressure. This effect is mimicked in the hindlimb unloaded rat model. After a duration of 8 days of unloading, Ca2+ signals activated by depolarization and inositol-1,4,5-trisphosphate intracellular release were increased in cerebral arteries. In the presence of ryanodine and thapsigargin, the depolarization-induced Ca2+ signals remained increased in hindlimb suspended animals, indicating that Ca2+ influx and Ca2+-induced Ca2+ release mechanism were both increased. Spontaneous Ca2+ waves and localized Ca2+ events were also investigated. Increases in both amplitude and frequency of spontaneous Ca2+ waves were measured in hindlimb suspension conditions. After pharmacological segregation of Ca2+ sparks and Ca2+ sparklets, their kinetic parameters were characterized. Hindlimb suspension induced an increase in the frequencies of both Ca2+ localized events, suggesting an increase of excitability. Labeling with bodipy compounds suggested that voltage-dependent Ca2+ channels and ryanodine receptor expressions were increased. Finally, the expression of the ryanodine receptor subtype 1 (RyR1) was increased in hindlimb unloading conditions. Taken together, these results suggest that RyR1 expression and voltage-dependent Ca2+ channels activity are the focal points of the regulation of Ca2+ signals activated by vasoconstriction in rat cerebral arteries with an increase of the voltage-dependent Ca2+ influx. PMID:24233561

  11. Crystal structures of ryanodine receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant

    NASA Astrophysics Data System (ADS)

    Yuchi, Zhiguang; Yuen, Siobhan M. Wong King; Lau, Kelvin; Underhill, Ainsley Q.; Cornea, Razvan L.; Fessenden, James D.; van Petegem, Filip

    2015-08-01

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca2+-dependent signalling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2-1.5 Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modelling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-binding protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding.

  12. Ryanodine receptors blockade reduces Amyloid-beta load and memory impairments in Tg2576 mouse model of Alzheimer disease

    PubMed Central

    Oulès, Bénédicte; Del Prete, Dolores; Greco, Barbara; Zhang, Xuexin; Lauritzen, Inger; Sevalle, Jean; Moreno, Sebastien; Paterlini-Bréchot, Patrizia; Trebak, Mohamed; Checler, Frédéric; Benfenati, Fabio; Chami, Mounia

    2012-01-01

    In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins (PS), the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP) thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP695), or APP harboring the Swedish double mutation (APPswe) triggers increased Ryanodine receptors (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches. PMID:22915123

  13. miRNAs regulated overexpression of ryanodine receptor is involved in chlorantraniliprole resistance in Plutella xylostella (L.)

    PubMed Central

    Li, Xiuxia; Guo, Lei; Zhou, Xuguo; Gao, Xiwu; Liang, Pei

    2015-01-01

    The amino acid mutations in ryanodine receptor (RyR) and elevated activity of detoxification enzymes have been associated with the diamide insecticide resistance in the diamondback moth, Plutella xylostella (L.). The up-regulation of P. xylostella RyR mRNA (PxRyR) expression has also been reported in field populations of different graphical origin. However, whether the up-regulation of PxRyR is involved in diamide resistance remains unknown. In this paper, 2.28- to 4.14-fold higher expression of PxRyR was detected in five field collected resistant populations, compared to that in a susceptible population. The expression of PxRyR was up-regulated 5.0- and 7.2-fold, respectively, after P. xylostella was treated with LC50 and LC75 of chlorantraniliprole for 12 h. Suppression of PxRyR using RNA interference restored the toxicity of chlorantraniliprole against the fourth instar larvae from the resistant population. More importantly, the expression of PxRyR is regulated by two miRNAs, miR-7a and miR-8519. These findings provide an empirical evidence of the involvement of miRNAs in the regulation of insecticide resistance, and shed light on the novel targets for the sustainable management of this devastating insect pest. PMID:26370154

  14. Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae.

    PubMed

    Troczka, B J; Williams, A J; Bass, C; Williamson, M S; Field, L M; Davies, T G E

    2015-02-10

    The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel. PMID:25447916

  15. Ryanodine receptor antagonists adapt NPC1 proteostasis to ameliorate lipid storage in Niemann-Pick type C disease fibroblasts.

    PubMed

    Yu, Ting; Chung, Chan; Shen, Dongbiao; Xu, Haoxing; Lieberman, Andrew P

    2012-07-15

    Niemann-Pick type C disease is a lysosomal storage disorder most often caused by loss-of-function mutations in the NPC1 gene. The encoded multipass transmembrane protein is required for cholesterol efflux from late endosomes and lysosomes. Numerous missense mutations in the NPC1 gene cause disease, including the prevalent I1061T mutation that leads to protein misfolding and degradation. Here, we sought to modulate the cellular proteostasis machinery to achieve functional recovery in primary patient fibroblasts. We demonstrate that targeting endoplasmic reticulum (ER) calcium levels using ryanodine receptor (RyR) antagonists increased steady-state levels of the NPC1 I1061T protein. These compounds also promoted trafficking of mutant NPC1 to late endosomes and lysosomes and rescued the aberrant storage of cholesterol and sphingolipids that is characteristic of disease. Similar rescue was obtained using three distinct RyR antagonists in cells with missense alleles, but not with null alleles, or by over-expressing calnexin, a calcium-dependent ER chaperone. Our work highlights the utility of proteostasis regulators to remodel the protein-folding environment in the ER to recover function in the setting of disease-causing missense alleles. PMID:22505584

  16. Crystal structures of Ryanodine Receptor SPRY1 and tandem-repeat domains reveal a critical FKBP12 binding determinant

    PubMed Central

    Yuchi, Zhiguang; Wong King Yuen, Siobhan M.; Lau, Kelvin; Underhill, Ainsley Q.; Cornea, Razvan L.; Fessenden, James D.; Van Petegem, Filip

    2015-01-01

    Ryanodine receptors (RyRs) form calcium release channels located in the membranes of the sarcoplasmic and endoplasmic reticulum. RyRs play a major role in excitation-contraction coupling and other Ca2+-dependent signaling events, and consist of several globular domains that together form a large assembly. Here we describe the crystal structures of the SPRY1 and tandem-repeat domains at 1.2 – 1.5Å resolution, which reveal several structural elements not detected in recent cryo-EM reconstructions of RyRs. The cryo-EM studies disagree on the position of SPRY domains, which had been proposed based on homology modeling. Computational docking of the crystal structures, combined with FRET studies, show that the SPRY1 domain is located next to FK506-Binding Protein (FKBP). Molecular dynamics flexible fitting and mutagenesis experiments suggest a hydrophobic cluster within SPRY1 that is crucial for FKBP binding. A RyR1 disease mutation, N760D, appears to directly impact FKBP binding through interfering with SPRY1 folding. PMID:26245150

  17. Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae

    PubMed Central

    Troczka, B.J.; Williams, A.J.; Bass, C.; Williamson, M.S.; Field, L.M.; Davies, T.G.E.

    2015-01-01

    The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel. PMID:25447916

  18. Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study.

    PubMed

    Stanczyk, Paulina J; Lai, F Anthony; Zissimopoulos, Spyros

    2016-01-01

    Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca(2+) release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins. PMID:27500320

  19. Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites.

    PubMed

    Gaburjakova, Jana; Gaburjakova, Marta

    2016-06-01

    A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca(2+) is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M(2+): Mg(2+), Ca(2+), Sr(2+) or Ba(2+)). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M(2+). Moreover, we performed competition experiments by varying the concentration of luminal M(2+) (Mg(2+), Sr(2+) or Ba(2+)) from 8mM to 53mM and investigated its ability to compete with 1mM luminal Ca(2+). We demonstrate that all tested M(2+) bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca(2+)>Sr(2+)>Mg(2+)~Ba(2+), showing a strong correlation with the M(2+) affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function. PMID:26849106

  20. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

    PubMed Central

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M. Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S. R. Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2. PMID:26405799

  1. The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.

    PubMed

    Xiao, Zhichao; Guo, Wenting; Yuen, Siobhan M Wong King; Wang, Ruiwu; Zhang, Lin; Van Petegem, Filip; Chen, S R Wayne

    2015-01-01

    The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1-547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2. PMID:26405799

  2. Cardiac calcium release channel (ryanodine receptor) in control and cardiomyopathic human hearts: mRNA and protein contents are differentially regulated.

    PubMed

    Sainte Beuve, C; Allen, P D; Dambrin, G; Rannou, F; Marty, I; Trouvé, P; Bors, V; Pavie, A; Gandgjbakch, I; Charlemagne, D

    1997-04-01

    Abnormal intracellular calcium handling in cardiomyopathic human hearts has been associated with an impaired function of the sarcoplasmic reticulum, but previous reports on the gene expression of the ryanodine receptors (Ry2) are contradictory. We measured the mRNA levels, the protein levels and the number of high affinity [3H]ryanodine binding sites in the left ventricle of non-failing (n = 9) and failing human hearts [idiopathic dilated (IDCM n = 16), ischemic (ICM n = 7) or mixed (MCM n = 8) cardiomyopathies]. Ry2 mRNA levels were significantly reduced in IDCM (-30%) and unchanged in MCM and ICM and Ry2 protein levels were similar. In contrast, we observed a two-fold increase in the number of high affinity Ry2 (B(max) = 0.43 +/- 0.11 v 0.22 +/- 0.13 pmol/mg protein, respectively; P<0.01) and an unchanged K(d). Furthermore, levels of myosin heavy chain mRNA and protein per g of tissue were similar in failing and non-failing hearts, suggesting that the observed differences in Ry2 are not caused by the increase in fibrosis in failing heart. Therefore, the dissociation between the two-fold increase in the number of high affinity ryanodine receptors observed in all failing hearts and the slightly decreased mRNA level or unchanged protein level suggests that the ryanodine binding properties are affected in failing myocardium and that such modifications rather than a change in gene expression alter the channel activity and could contribute to abnormalities in intracellular Ca2+ handling. PMID:9160875

  3. Homeostatic and stimulus-induced coupling of the L-type Ca2+ channel to the ryanodine receptor in the hippocampal neuron in slices

    PubMed Central

    Berrout, Jonathan; Isokawa, Masako

    2009-01-01

    Activity-dependent increase in cytosolic calcium ([Ca2+]i) is a prerequisite for many neuronal functions. We previously reported a strong direct depolarization, independent of glutamate receptors, effectively caused a release of Ca2+ from ryanodine sensitive stores and induced the synthesis of endogenous cannabinoids (eCBs) and eCB-mediated responses. However, the cellular mechanism that initiated the depolarization-induced Ca2+ release is not completely understood. In the present study, we optically recorded [Ca2+]i from CA1 pyramidal neurons in the hippocampal slice and directly monitored miniature Ca2+ activities and depolarization-induced Ca2+ signals in order to determine the source(s) and properties of [Ca2+]i-dynamics that could lead to a release of Ca2+ from the ryanodine receptor. In the absence of depolarizing stimuli, spontaneously-occurring miniature Ca2+ events were detected from a group of hippocampal neurons. This miniature Ca2+ event persisted in the nominal Ca2+-containing artificial cerebrospinal fluid (ACSF), and increased in frequency in response to the bath-application of caffeine and KCl. In contrast, nimodipine, the antagonist of the L-type Ca2+ channel (LTCC), a high concentration of ryanodine, the antagonist of the ryanodine receptor (RyR), and thapsigargin (TG) reduced the occurrence of the miniature Ca2+ events. When a brief puff-application of KCl was given locally to the soma of individual neurons in the presence of glutamate receptor antagonists, these neurons generated a transient increase in the [Ca2+]i in the dendrosomal region. This [Ca2+]i-transient was sensitive to nimodipine, TG, and ryanodine suggesting that the [Ca2+]i-transient was caused primarily by the LTCC-mediated Ca2+-influx and a release of Ca2+ from RyR. We observed little contribution from N-or P/Q-type Ca2+ channels. The coupling between LTCC and RyR was direct and independent of synaptic activities. Immunohistochemical study revealed a cellular localization of LTCC

  4. Molecular Characteristics, mRNA Expression, and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  5. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer

    PubMed Central

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-01-01

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed. PMID:26573433

  6. Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise-induced sudden cardiac death.

    PubMed

    Terentyev, Dmitry; Nori, Alessandra; Santoro, Massimo; Viatchenko-Karpinski, Serge; Kubalova, Zuzana; Gyorke, Inna; Terentyeva, Radmila; Vedamoorthyrao, Srikanth; Blom, Nico A; Valle, Giorgia; Napolitano, Carlo; Williams, Simon C; Volpe, Pompeo; Priori, Silvia G; Gyorke, Sandor

    2006-05-12

    Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmogenic disorder associated with mutations in the cardiac ryanodine receptor (RyR2) and cardiac calsequestrin (CASQ2) genes. Previous in vitro studies suggested that RyR2 and CASQ2 interact as parts of a multimolecular Ca(2+)-signaling complex; however, direct evidence for such interactions and their potential significance to myocardial function remain to be determined. We identified a novel CASQ2 mutation in a young female with a structurally normal heart and unexplained syncopal episodes. This mutation results in the nonconservative substitution of glutamine for arginine at amino acid 33 of CASQ2 (R33Q). Adenoviral-mediated expression of CASQ2(R33Q) in adult rat myocytes led to an increase in excitation-contraction coupling gain and to more frequent occurrences of spontaneous propagating (Ca2+ waves) and local Ca2+ signals (sparks) with respect to control cells expressing wild-type CASQ2 (CASQ2WT). As revealed by a Ca2+ indicator entrapped inside the sarcoplasmic reticulum (SR) of permeabilized myocytes, the increased occurrence of spontaneous Ca2+ sparks and waves was associated with a dramatic decrease in intra-SR [Ca2+]. Recombinant CASQ2WT and CASQ2R33Q exhibited similar Ca(2+)-binding capacities in vitro; however, the mutant protein lacked the ability of its WT counterpart to inhibit RyR2 activity at low luminal [Ca2+] in planar lipid bilayers. We conclude that the R33Q mutation disrupts interactions of CASQ2 with the RyR2 channel complex and impairs regulation of RyR2 by luminal Ca2+. These results show that intracellular Ca2+ cycling in normal heart relies on an intricate interplay of CASQ2 with the proteins of the RyR2 channel complex and that disruption of these interactions can lead to cardiac arrhythmia. PMID:16601229

  7. Simulation of the effect of rogue ryanodine receptors on a calcium wave in ventricular myocytes with heart failure

    NASA Astrophysics Data System (ADS)

    Lu, Luyao; Xia, Ling; Ye, Xuesong; Cheng, Heping

    2010-06-01

    Calcium homeostasis is considered to be one of the most important factors for the contraction and relaxation of the heart muscle. However, under some pathological conditions, such as heart failure (HF), calcium homeostasis is disordered, and spontaneous waves may occur. In this study, we developed a mathematical model of formation and propagation of a calcium wave based upon a governing system of diffusion-reaction equations presented by Izu et al (2001 Biophys. J. 80 103-20) and integrated non-clustered or 'rogue' ryanodine receptors (rogue RyRs) into a two-dimensional (2D) model of ventricular myocytes isolated from failing hearts in which sarcoplasmic reticulum (SR) Ca2+ pools are partially unloaded. The model was then used to simulate the effect of rogue RyRs on initiation and propagation of the calcium wave in ventricular myocytes with HF. Our simulation results show that rogue RyRs can amplify the diastolic SR Ca2+ leak in the form of Ca2+ quarks, increase the probability of occurrence of spontaneous Ca2+ waves even with smaller SR Ca2+ stores, accelerate Ca2+ wave propagation, and hence lead to delayed afterdepolarizations (DADs) and cardiac arrhythmia in the diseased heart. This investigation suggests that incorporating rogue RyRs in the Ca2+ wave model under HF conditions provides a new view of Ca2+ dynamics that could not be mimicked by adjusting traditional parameters involved in Ca2+ release units and other ion channels, and contributes to understanding the underlying mechanism of HF.

  8. Exome Sequencing Reveals Novel Rare Variants in the Ryanodine Receptor and Calcium Channel Genes in Malignant Hyperthermia Families

    PubMed Central

    Kim, Jerry H.; Jarvik, Gail P.; Browning, Brian L.; Rajagopalan, Ramakrishnan; Gordon, Adam S.; Rieder, Mark J.; Robertson, Peggy D.; Nickerson, Deborah A.; Fisher, Nickla A.; Hopkins, Philip M.

    2014-01-01

    Background About half of malignant hyperthermia (MH) cases are associated with skeletal muscle ryanodine receptor 1 (RYR1) and calcium channel, voltage-dependent, L type, α1S subunit (CACNA1S) gene mutations, leaving many with an unknown cause. We chose to apply a sequencing approach to uncover causal variants in unknown cases. Sequencing the exome, the protein-coding region of the genome, has power at low sample sizes and identified the cause of over a dozen Mendelian disorders. Methods We considered four families with multiple MH cases but in whom no mutations in RYR1 and CACNA1S had been identified by Sanger sequencing of complementary DNA. Exome sequencing of two affecteds per family, chosen for maximum genetic distance, were compared. Variants were ranked by allele frequency, protein change, and measures of conservation among mammals to assess likelihood of causation. Finally, putative pathogenic mutations were genotyped in other family members to verify cosegregation with MH. Results Exome sequencing revealed 1 rare RYR1 nonsynonymous variant in each of 3 families (Asp1056His, Val2627Met, Val4234Leu), and 1 CACNA1S variant (Thr1009Lys) in a 4th family. These were not seen in variant databases or in our control population sample of 5379 exomes. Follow-up sequencing in other family members verified cosegregation of alleles with MH. Conclusions Using both exome sequencing and allele frequency data from large sequencing efforts may aid genetic diagnosis of MH. In our sample, it was more sensitive for variant detection in known genes than Sanger sequencing of complementary DNA, and allows for the possibility of novel gene discovery. PMID:24013571

  9. FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6.

    PubMed

    Galfré, Elena; Pitt, Samantha J; Venturi, Elisa; Sitsapesan, Mano; Zaccai, Nathan R; Tsaneva-Atanasova, Krasimira; O'Neill, Stephen; Sitsapesan, Rebecca

    2012-01-01

    Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias. PMID:22363773

  10. Cardiac ryanodine receptor activation by a high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

    PubMed Central

    Hanna, Amy D.; Lam, Alex; Thekkedam, Chris; Gallant, Esther M.; Beard, Nicole A.; Dulhunty, Angela F.

    2014-01-01

    ABSTRACT Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP or buffering of the redox potential to an oxidising value (−180 mV), RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm. PMID:25146393

  11. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise.

    PubMed

    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Ruas, Jorge L; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T; Skurvydas, Albertas; Westerblad, Håkan

    2015-12-15

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca(2+) release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca(2+) leak at rest, and depressed force production due to impaired SR Ca(2+) release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca(2+)-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group. PMID:26575622

  12. Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

    PubMed Central

    Place, Nicolas; Ivarsson, Niklas; Venckunas, Tomas; Neyroud, Daria; Brazaitis, Marius; Cheng, Arthur J.; Ochala, Julien; Kamandulis, Sigitas; Girard, Sebastien; Volungevičius, Gintautas; Paužas, Henrikas; Mekideche, Abdelhafid; Kayser, Bengt; Martinez-Redondo, Vicente; Bruton, Joseph; Truffert, Andre; Lanner, Johanna T.; Skurvydas, Albertas; Westerblad, Håkan

    2015-01-01

    High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca2+ leak at rest, and depressed force production due to impaired SR Ca2+ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca2+-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group. PMID:26575622

  13. Molecular characterization of human thyroid hormone receptor β isoform 4.

    PubMed

    Moriyama, Kenji; Yamamoto, Hiroyuki; Futawaka, Kumi; Atake, Asami; Kasahara, Masato; Tagami, Tetsuya

    2016-01-01

    Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus. PMID:26513165

  14. Splice isoform estrogen receptors as integral transmembrane proteins

    PubMed Central

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R.

    2011-01-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus–truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element–luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets. PMID:21937726

  15. Role of nuclear progesterone receptor isoforms in uterine pathophysiology

    PubMed Central

    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    BACKGROUND Progesterone is a key hormonal regulator of the female reproductive system. It plays a major role to prepare the uterus for implantation and in the establishment and maintenance of pregnancy. Actions of progesterone on the uterine tissues (endometrium, myometrium and cervix) are mediated by the combined effects of two progesterone receptor (PR) isoforms, designated PR-A and PR-B. Both receptors function primarily as ligand-activated transcription factors. Progesterone action on the uterine tissues is qualitatively and quantitatively determined by the relative levels and transcriptional activities of PR-A and PR-B. The transcriptional activity of the PR isoforms is affected by specific transcriptional coregulators and by PR post-translational modifications that affect gene promoter targeting. In this context, appropriate temporal and cell-specific expression and function of PR-A and PR-B are critical for normal uterine function. METHODS Relevant studies describing the role of PRs in uterine physiology and pathology (endometriosis, uterine leiomyoma, endometrial cancer, cervical cancer and recurrent pregnancy loss) were comprehensively searched using PubMed, Cochrane Library, Web of Science, and Google Scholar and critically reviewed. RESULTS Progesterone, acting through PR-A and PR-B, regulates the development and function of the endometrium and induces changes in cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs promotes myometrial relaxation and cervical closure. Withdrawal of PR-mediated progesterone signaling triggers menstruation and parturition. PR-mediated progesterone signaling is anti-mitogenic in endometrial epithelial cells, and as such, mitigates the tropic effects of estrogen on eutopic normal endometrium, and on ectopic implants in endometriosis. Similarly, ligand-activated PRs function as tumor suppressors in endometrial cancer cells through inhibition of key

  16. Participation of inositol trisphosphate and ryanodine receptors in Bufo arenarum oocyte activation.

    PubMed

    Ajmat, M T; Bonilla, F; Zelarayán, L; Bühler, M I

    2011-05-01

    Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear. PMID:20880424

  17. Reversal in Cognition Impairments, Cholinergic Dysfunction, and Cerebral Oxidative Stress Through the Modulation of Ryanodine Receptors (RyRs) and Cysteinyl Leukotriene-1 (CysLT1) Receptors.

    PubMed

    Singh, Prabhat; Sharma, Bhupesh

    2016-01-01

    Chronic cerebral hypoperfusion (CCH) is a general pathophysiological condition occurring in vascular dementia (VaD) associated with negative impact on cognitive functions. Ryanodine as well as cysteinyl leukotriene-1 receptors (RyRs and CysLT1Rs) are extensively present in the central nervous system, where they participate in regulation of cognition, motivation, inflammation and neurodegeneration. The purpose of this study is to examine the role of ruthenium red; a selective RyR blocker as well as montelukast; a specific CysLT1 antagonist in CCH induced VaD in mice. Two vessel occlusion (2VO) or permanent ligation of bilateral common carotid arteries technique was used to induce CCH in mice. Animals with bilateral carotid arteries occlusion have revealed impaired learning and memory (Morris water maze), cholinergic dysfunction (increased acetylcholinesterase activity) as well as increased brain oxidative stress (reduction in brain superoxide dismutase, glutathione and catalase with an increase in thiobarbituric acid reactive substance level), with increased brain infarct size (2,3,5-triphenylterazolium chloride staining). While, administration of ruthenium red and montelukast considerably attenuated CCH induced cognitive impairments, cholinergic dysfunction, brain oxidative stress as well as brain damage. The results suggest that bilateral carotid arteries occlusion induced CCH has brought out VaD, which was attenuated by treatment with ruthenium red and montelukast. Therefore, modulation of RyRs as well as CysLT1 receptors may provide help in conditions involving CCH such as cognitive impairment and VaD. PMID:26500103

  18. Involvement of plasma membrane Ca2+ channels, IP3 receptors, and ryanodine receptors in the generation of spontaneous rhythmic contractions of the cricket lateral oviduct.

    PubMed

    Tamashiro, Hirotake; Yoshino, Masami

    2014-12-01

    In the present study, the isolated cricket (Gryllus bimaculatus) lateral oviduct exhibited spontaneous rhythmic contractions (SRCs) with a frequency of 0.29±0.009 Hz (n=43) and an amplitude of 14.6±1.25 mg (n=29). SRCs completely disappeared following removal of extracellular Ca2+ using a solution containing 5mM EGTA. Application of the non-specific Ca2+ channel blockers Co2+, Ni2+, and Cd2+ also decreased both the frequency and amplitude of SRCs in dose-dependent manners, suggesting that Ca2+ entry through plasma membrane Ca2+ channels is essential for the generation of SRCs. Application of ryanodine (30 μM), which depletes intracellular Ca2+ by locking ryanodine receptor (RyR)-Ca2+ channels in an open state, gradually reduced the frequency and amplitude of SRCs. A RyR antagonist, tetracaine, reduced both the frequency and amplitude of SRCs, whereas a RyR activator, caffeine, increased the frequency of SRCs with a subsequent increase in basal tonus, indicating that RyRs are essential for generating SRCs. To further investigate the involvement of phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in SRCs, we examined the effect of a PLC inhibitor, U73122, and an IP3R antagonist, 2-aminoethoxydiphenyl borate (2-APB), on SRCs. Separately, U73122 (10 μM) and 2-APB (30-50 μM) both significantly reduced the amplitude of SRCs with little effect on their frequency, further indicating that the PLC/IP3R signaling pathway is fundamental to the modulation of the amplitude of SRCs. A hypotonic-induced increase in the frequency and amplitude of SRCs and a hypertonic-induced decrease in the frequency and amplitude of SRCs indicated that mechanical stretch of the lateral oviduct is involved in the generation of SRCs. The sarcoplasmic reticulum Ca2+-pump ATPase inhibitors thapsigargin and cyclopiazonic acid impaired or suppressed the relaxation phase of SRCs. Taken together, the present results indicate that Ca2+ influx through plasma membrane Ca2

  19. Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling.

    PubMed

    Wescott, Andrew P; Jafri, M Saleet; Lederer, W J; Williams, George S B

    2016-03-01

    Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for

  20. Molecular characterization of a ryanodine receptor gene in the rice leaffolder, Cnaphalocrocis medinalis (Guenée).

    PubMed

    Wang, Jianjun; Li, Yanqing; Han, Zhaojun; Zhu, Youli; Xie, Zhijuan; Wang, Jian; Liu, Yaping; Li, Xianchun

    2012-01-01

    Ryanodine receptors (RyRs) are the targets of two novel classes of synthetic insecticidal chemicals, phthalic acid diamides and anthranilic diamides. Isolation of full-length RyR cDNAs is a critical step towards the structural and functional characterization of insect RyRs and an understanding of the molecular mechanisms underlying the species selective toxicity of diamide insecticides. However, there has been little research on the insect RyR genes due to the high molecular weight of the RyR proteins. In this study, we isolated a full-length RyR cDNA (named as CmRyR) from Cnaphalocrocis medinalis, an important rice pest throughout Southeast Asia. The composite CmRyR gene contains an ORF of 15264 bp encoding a protein of 5087 amino acid residues, which shares 79% overall identity with its Drosophila melanogaster homologue. All hallmarks of the RyR proteins are conserved in the CmRyR protein, suggesting that CmRyR is a structural and functional analogue of known RyRs. A multiple sequence alignment illustrates that the insect RyRs share high levels of amino acid sequence identity at the the COOH-terminal region. However, the amino acid residues analogous to the CmRyR residues N(4922), N(4924), N(4935), L(4950), L(4981), N(5013) and T(5064) are unique to lepidopteran RyRs compared with non-lepidopteran insect RyRs. This finding suggests that these residues may be involved in the differences in channel properties between lepidopteran and non-lepidopteran insect RyRs and in the species selective toxicity of diamide insecticides. Furthermore, two alternative splicing sites were identified in the CmRyR gene, one of which was located in the central part of the predicted second SPRY domain. Diagnostic PCR showed that the inclusion frequencies of two mutually exclusive exons (a/b) and one optional exon (c) differed between developmental stages or adult anatomical regions. Our results imply that alternative splicing may be a major means of generating functional diversity in C

  1. 5-HT2B receptor-mediated calcium release from ryanodine-sensitive intracellular stores in human pulmonary artery endothelial cells.

    PubMed Central

    Ullmer, C.; Boddeke, H. G.; Schmuck, K.; Lübbert, H.

    1996-01-01

    1. We have characterized the 5-hydroxytryptamine (5-HT)-induced calcium signalling in endothelial cells from the human pulmonary artery. Using RT-PCR we show, that of all cloned G-protein coupled 5-HT receptors, these cells express only 5-HT1D beta, 5-HT2B and little 5-HT4 receptor mRNA. 2. In endothelial cells 5-HT inhibits the formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) via 5-HT1D beta receptors but fails to activate phosphoinositide (PI) turnover. However, the latter pathway is strongly activated by histamine. 3. Despite the lack of detectable inositol phosphate (IP) formation in human pulmonary artery endothelial cells, 5-HT (pD2 = 5.82 +/- 0.06, n = 6) or the selective 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (pD2 = 5.66 +/- 0.03, n = 7) elicited transient calcium signals comparable to those evoked by histamine (pD2 = 6.44 +/- 0.01, n = 7). Since 5-HT2A and 5-HT2C receptor mRNAs are not detectable in pulmonary artery endothelial cells, activation of 5-HT2B receptors is responsible for the transient calcium release. The calcium transients are independent of the inhibition of adenylate cyclase, since DOI does not stimulate 5-HT1D beta receptors. 4. Both, the 5-HT- and histamine-stimulated calcium signals were also observed when the cells were placed in calcium-free medium. This indicates that 5-HT triggers calcium release from intracellular stores. 5. Heparin is an inhibitor of the IP3-activated calcium release channels on the endoplasmic reticulum. Intracellular infusion of heparin through patch pipettes in voltage clamp experiments failed to block 5-HT-induced calcium signals, whereas it abolished the histamine response. This supports the conclusion that the 5-HT-induced calcium release is independent of IP3 formation. 6. Unlike the histamine response, the 5-HT response was sensitive to micromolar concentrations of ryanodine and, to a lesser extent, ruthenium red. This implies that 5-HT2B receptors trigger calcium

  2. Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

    PubMed Central

    Wang, Jian; Shimoda, Larissa A.

    2012-01-01

    In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca2+ concentration ([Ca2+]i) by inducing Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx through store- and voltage-operated Ca2+ channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca2+ release, we measured [Ca2+]i with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP3R), respectively, or 4% O2 caused rapid transient increases in [Ca2+]i, indicating intracellular Ca2+ release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca2+-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca2+ release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca2+ release to caffeine and hypoxia but not norepinephrine. The IP3R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca2+ release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca2+]i that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca2+]i induced by hypoxia, as well as the subsequent Ca2+ influx that sustained this increase, required release of Ca2+ from both RyR and IP3R, and 2) the SR Ca2+ stores accessed by RyR, IP3R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca2+-ATPase. PMID:22582116

  3. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

    PubMed

    Mundell, Stuart J; Jones, Matthew L; Hardy, Adam R; Barton, Johanna F; Beaucourt, Stephanie M; Conley, Pamela B; Poole, Alastair W

    2006-09-01

    ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking. PMID:16804093

  4. Membrane depolarization increases ryanodine sensitivity to Ca2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation-contraction coupling.

    PubMed

    Pitake, Saumitra; Ochs, Raymond S

    2016-04-01

    The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation-contraction coupling. However, the mechanism for subsequent Ca(2+) release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca(2+) under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation-contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca(2+) from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca(2+) was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca(2+) concentration in the media. Ca(2+) entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca(2+) entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca(2+) release from the ryanodine receptor to the cytosol. PMID:26643865

  5. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes

    PubMed Central

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  6. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

    PubMed

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  7. Crystal structure of type I ryanodine receptor amino-terminal beta-trefoil domain reveals a disease-associated mutation "hot spot" loop.

    PubMed

    Amador, Fernando J; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J; Wilson, Aaron; MacLennan, David H; Ikura, Mitsuhiko

    2009-07-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca(2+). Ca(2+) is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca(2+) transient by activation of a class of Ca(2+) release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca(2+)-ATPases (SERCAs) to terminate the Ca(2+) transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR(NTD)) at 2.5 A. The RyR(NTD) structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP(3)Rsup), but lacks most of the long helix-turn-helix segment of the "arm" domain in IP(3)Rsup. The N-terminal beta-trefoil fold, found in both RyR and IP(3)R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation "hot spot" loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyR(NTD), supporting previously described mechanisms whereby mutations perturb protein-protein interactions. PMID:19541610

  8. Crystal structure of type I ryanodine receptor amino-terminal [beta]-trefoil domain reveals a disease-associated mutation 'hot spot' loop

    SciTech Connect

    Amador, Fernando J.; Liu, Shuang; Ishiyama, Noboru; Plevin, Michael J.; Wilson, Aaron; MacLennan, David H.; Ikura, Mitsuhiko

    2009-12-01

    Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca{sup 2+}. Ca{sup 2+} is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca{sup 2+} transient by activation of a class of Ca{sup 2+} release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca{sup 2+}-ATPases (SERCAs) to terminate the Ca{sup 2+} transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR{sub NTD}) at 2.5 {angstrom}. The RyR{sub NTD} structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP3Rsup), but lacks most of the long helix-turn-helix segment of the 'arm' domain in IP3Rsup. The N-terminal {beta}-trefoil fold, found in both RyR and IP{sub 3}R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation 'hot spot' loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyRNTD, supporting previously described mechanisms whereby mutations perturb protein-protein interactions.

  9. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

    PubMed Central

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  10. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy).

    PubMed

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  11. Immunohistochemical localization of progesterone receptor isoforms and estrogen receptor alpha in the chicken oviduct magnum during development.

    PubMed

    González-Morán, María Genoveva

    2015-10-01

    In this work, the immunohistochemical expression of progesterone receptor (PR) isoforms and estrogen receptor alpha (ER-α), as well as the histomorphometric changes of the magnum region of the left oviduct from 8-day-old chicken embryos to one-month-old chickens were evaluated. Results indicate evident histological changes in the oviduct magnum during development mainly in the magnum's mucosa. Immunohistochemical analysis showed that the oviduct magnum from 8-day-old chicken embryos to one-day-old chickens did not present any PR isoform, but the oviduct magnum of one-week and one-month-old chickens expressed PR in the nuclei of all cell types. In epithelial cells, PR-B was the only isoform expressed; in muscle and serosa cells, PR-A isoform was the only isoform expressed; and stromal cells expressed both isoforms. The results also demonstrate positive ER-α immunostaining in the nuclei of different cells from embryonic life to later developmental stages of the oviduct magnum. Data indicate that the variations of ER-α or PR expression or dominance of either PR expression is differentially regulated depending on the cell type, the development of the oviduct, and in an age-specific manner. These variations in sex steroids hormone receptors are related with histological changes of the oviduct magnum through development. PMID:26519127

  12. Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2

    PubMed Central

    Gauthier, Sebastien A.; VanHaaften, Eric; Cherbas, Lucy; Cherbas, Peter; Hewes, Randall S.

    2012-01-01

    The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators. PMID:22912598

  13. Expression of leptin and leptin receptor isoforms in the rat and human carotid body.

    PubMed

    Porzionato, Andrea; Rucinski, Marcin; Macchi, Veronica; Stecco, Carla; Castagliuolo, Ignazio; Malendowicz, Ludwik K; De Caro, Raffaele

    2011-04-18

    Leptin is known to play a role in the modulation of metabolism and control of breathing acting mainly on central nervous structures, although additional actions on peripheral arterial chemoreceptors have also been suggested in the literature. We therefore examined by means of immunohistochemistry the expression of leptin and leptin receptors in the carotid bodies of rats and humans. Leptin expression and relative expression of leptin receptor isoforms were also studied in rats by real-time PCR. No leptin or leptin receptor immunoreactivities were visible in the type II cells of either series. In rat carotid bodies, diffuse positive stainings for leptin and leptin receptors (both with antibody recognizing all receptor isoforms and antibody specific for Ob-Rb) were observed in type I cells. In human carotid bodies, the mean percentage (±standard error) of leptin immunoreactive type I cells was 39.4%±5.1% and the percentages of leptin receptor immunoreactive type I cells were 57.3%±3.9% with antibody recognizing all receptor isoforms and 33.3%±4.2% with antibody specific for isoform Ob-Rb. Double immunofluorescences with anti-tyrosine hydroxylase (type I cell marker) and anti-glial fibrillary acidic protein (type II cell markers) confirmed the selective location of leptin and Ob-Rb in type I cells. Real-time PCR showed the expression of leptin and Ob-Ra, Ob-Rb, Ob-Rc and Ob-Rf isoform mRNA in the rat carotid body, levels of expression being Ob-Rf>Ob-Rc>Ob-Ra>Ob-Rb. Ob-Re mRNA was not detected. The above findings suggest a role of circulating or locally produced leptin in the regulation of chemoreceptor discharge and/or metabolic sensing function, by means of direct action on type I cells. PMID:21334312

  14. Multiple actions of φ-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins

    PubMed Central

    Smith, Jennifer J.; Vetter, Irina; Lewis, Richard J.; Peigneur, Steve; Tytgat, Jan; Lam, Alexander; Gallant, Esther M.; Beard, Nicole A.; Alewood, Paul F.; Dulhunty, Angela F.

    2013-01-01

    We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as φ-liotoxin-Lw1a (φ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of φ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. φ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia. PMID:23671114

  15. Nitric Oxide-Induced Calcium Release: Activation of Type 1 Ryanodine Receptor, a Calcium Release Channel, through Non-Enzymatic Post-Translational Modification by Nitric Oxide

    PubMed Central

    Kakizawa, Sho

    2013-01-01

    Nitric oxide (NO) is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3′, 5′-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: “S-nitrosylation” of target proteins. S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH), and categorized into non-enzymatic post-translational modification (PTM) of proteins, contrasted to enzymatic PTM of proteins, such as phosphorylation mediated by various protein kinases. Very recently, we found novel intracellular calcium (Ca2+) mobilizing mechanism, NO-induced Ca2+ release (NICR) in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1), a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic PTM of proteins by gaseous signals, are described. PMID:24130553

  16. Maternal high-altitude hypoxia and suppression of ryanodine receptor-mediated Ca2+ sparks in fetal sheep pulmonary arterial myocytes.

    PubMed

    Hadley, Scott R; Blood, Quintin; Rubalcava, Monica; Waskel, Edith; Lumbard, Britney; Le, Petersen; Longo, Lawrence D; Buchholz, John N; Wilson, Sean M

    2012-11-01

    Ca(2+) sparks are fundamental Ca(2+) signaling events arising from ryanodine receptor (RyR) activation, events that relate to contractile and dilatory events in the pulmonary vasculature. Recent studies demonstrate that long-term hypoxia (LTH) can affect pulmonary arterial reactivity in fetal, newborn, and adult animals. Because RyRs are important to pulmonary vascular reactivity and reactivity changes with ontogeny and LTH we tested the hypothesis that RyR-generated Ca(2+) signals are more active before birth and that LTH suppresses these responses. We examined these hypotheses by performing confocal imaging of myocytes in living arteries and by performing wire myography studies. Pulmonary arteries (PA) were isolated from fetal, newborn, or adult sheep that lived at low altitude or from those that were acclimatized to 3,801 m for > 100 days. Confocal imaging demonstrated preservation of the distance between the sarcoplasmic reticulum, nucleus, and plasma membrane in PA myocytes. Maturation increased global Ca(2+) waves and Ca(2+) spark activity, with sparks becoming larger, wider, and slower. LTH preferentially depressed Ca(2+) spark activity in immature pulmonary arterial myocytes, and these sparks were smaller, wider, and slower. LTH also suppressed caffeine-elicited contraction in fetal PA but augmented contraction in the newborn and adult. The influence of both ontogeny and LTH on RyR-dependent cell excitability shed new light on the therapeutic potential of these channels for the treatment of pulmonary vascular disease in newborns as well as adults. PMID:22962012

  17. Estrogen receptor isoforms and progestin hormone dependence in a mouse mammary tumor model.

    PubMed

    Actis, A M; Caruso, S P; Levin, E

    1994-09-01

    The close interaction between receptors and other transcription factors suggests that their corresponding transducing signals can trigger functional and structural changes in other related molecules. The effect of a progestinic agent, medroxyprogesterone acetate (MPA), on some of the estrogen-receptor (ER) parameters was studied in 2 murine mammary tumor sublines with different progestin hormone dependence for their respective growth. The relative binding affinity of estradiol and tamoxifen for the ER, the receptor content and the ER isoforms studied by HPLC were determined in the hormone-autonomous (HA) and the hormone-dependent (HD) tumor sublines. In the HA subline administration of MPA did not modify the tumor growth rate, whereas this was accelerated in the HD subline. The ER content was clearly increased in the HD tumor subline, but not in the HA subline, compared with the untreated controls. In contrast, the E2 and tamoxifen relative binding affinity for the ER and the isoform profiles were affected by MPA treatment in the HA, but not in the HD tumor subline. The functional change (decrease in relative binding affinity) can be attributed to the appearance of a lower-molecular-size ER isoform under the progestinic treatment. Modifications in one receptor molecule by the action of ligands corresponding to another type of receptor show the interconection between transcription factors and the necessity of broadening conventional concepts regarding hormone dependence in mammary tumorigenesis. PMID:8077051

  18. Expression and function of ryanodine receptor related pathways in PCB tolerant Atlantic killifish (Fundulus heteroclitus) from New Bedford Harbor, MA, USA

    PubMed Central

    Fritsch, Erika B.; Stegeman, John J.; Goldstone, Jared V.; Nacci, Diane E.; Champlin, Denise; Jayaraman, Saro; Connon, Richard E.; Pessah, Isaac N.

    2015-01-01

    Atlantic killifish (Fundulus heteroclitus) thrive in New Bedford Harbor (NBH), MA, highly contaminated with polychlorinated biphenyls (PCBs). Resident killifish have evolved tolerance to dioxin-like (DL) PCBs, whose toxic effects through the aryl hydrocarbon receptor (AhR) are well studied. In NBH, non-dioxin like PCBs (NDL PCBs), which lack activity toward the AhR, vastly exceed levels of DL congeners yet how killifish counter NDL toxic effects has not been explored. In mammals and fish, NDL PCBs are potent activators of ryanodine receptors (RyR), Ca2+ release channels necessary for a vast array of physiological processes. In the current study we compared the expression and function of RyR related pathways in NBH killifish with killifish from the reference site at Scorton Creek (SC, MA). Relative to the SC fish, adults from NBH displayed increased levels of skeletal muscle RyR1 protein, and increased levels of FK506-binding protein 12 kDa (FKBP12), an accessory protein essential for NDL PCB-triggered changes in RyR channel function. In accordance with increased RyR1 levels, NBH killifish displayed increased maximal ligand binding, increased maximal response to Ca2+ activation and increased maximal response to activation by the NDL PCB congener PCB 95. Compared to SC, NBH embryos and larvae had increased levels of mtor and ryr2 transcripts at multiple stages of development, and generations, while levels of serca2 were decreased at 9 days post-fertilization in the F1 and F2 generations. These findings suggest that there are compensatory and heritable changes in RyR mediated Ca2+ signaling proteins or potential signaling partners in NBH killifish. PMID:25546006

  19. Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain

    PubMed Central

    Vervliet, Tim; Lemmens, Irma; Vandermarliere, Elien; Decrock, Elke; Ivanova, Hristina; Monaco, Giovanni; Sorrentino, Vincenzo; Kasri, Nael Nadif; Missiaen, Ludwig; Martens, Lennart; De Smedt, Humbert; Leybaert, Luc; Parys, Jan B.; Tavernier, Jan; Bultynck, Geert

    2015-01-01

    Anti-apoptotic B-cell lymphoma 2 (Bcl-2) family members target several intracellular Ca2+-transport systems. Bcl-2, via its N-terminal Bcl-2 homology (BH) 4 domain, inhibits both inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs), while Bcl-XL, likely independently of its BH4 domain, sensitizes IP3Rs. It remains elusive whether Bcl-XL can also target and modulate RyRs. Here, Bcl-XL co-immunoprecipitated with RyR3 expressed in HEK293 cells. Mammalian protein-protein interaction trap (MAPPIT) and surface plasmon resonance (SPR) showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain, although to a lesser extent compared to the BH4 domain of Bcl-2. Consistent with the ability of the BH4 domain of Bcl-XL to bind to RyRs, loading the BH4-Bcl-XL peptide into RyR3-overexpressing HEK293 cells or in rat hippocampal neurons suppressed RyR-mediated Ca2+ release. In silico superposition of the 3D-structures of Bcl-2 and Bcl-XL indicated that Lys87 of the BH3 domain of Bcl-XL could be important for interacting with RyRs. In contrast to Bcl-XL, the Bcl-XLK87D mutant displayed lower binding affinity for RyR3 and a reduced inhibition of RyR-mediated Ca2+ release. These data suggest that Bcl-XL binds to RyR channels via its BH4 domain, but also its BH3 domain, more specific Lys87, contributes to the interaction. PMID:25872771

  20. BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms.

    PubMed

    Gassmann, Kathrin; Schreiber, Timm; Dingemans, Milou M L; Krause, Guido; Roderigo, Claudia; Giersiefer, Susanne; Schuwald, Janette; Moors, Michaela; Unfried, Klaus; Bergman, Åke; Westerink, Remco H S; Rose, Christine R; Fritsche, Ellen

    2014-08-01

    Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 μM) and 6-OH-BDE-47 (0.2 μM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment. PMID:24599297

  1. Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus.

    PubMed

    Westermeier, F; Sáez, T; Arroyo, P; Toledo, F; Gutiérrez, J; Sanhueza, C; Pardo, F; Leiva, A; Sobrevia, L

    2016-05-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26431063

  2. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells

    PubMed Central

    Chakedis, Jeffery; French, Randall; Babicky, Michele; Jaquish, Dawn; Howard, Haleigh; Mose, Evangeline; Lam, Raymond; Holman, Patrick; Miyamoto, Jaclyn; Walterscheid, Zakk; Lowy, Andrew M.

    2015-01-01

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription (IPT) domain. The splice variant is detected in 73% of pancreatic adenocarcinoma patient derived xenografts and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirms translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing (HPNE) cells activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  3. A novel protein isoform of the RON tyrosine kinase receptor transforms human pancreatic duct epithelial cells.

    PubMed

    Chakedis, J; French, R; Babicky, M; Jaquish, D; Howard, H; Mose, E; Lam, R; Holman, P; Miyamoto, J; Walterscheid, Z; Lowy, A M

    2016-06-23

    The MST1R gene is overexpressed in pancreatic cancer producing elevated levels of the RON tyrosine kinase receptor protein. While mutations in MST1R are rare, alternative splice variants have been previously reported in epithelial cancers. We report the discovery of a novel RON isoform discovered in human pancreatic cancer. Partial splicing of exons 5 and 6 (P5P6) produces a RON isoform that lacks the first extracellular immunoglobulin-plexin-transcription domain. The splice variant is detected in 73% of xenografts derived from pancreatic adenocarcinoma patients and 71% of pancreatic cancer cell lines. Peptides specific to RON P5P6 detected in human pancreatic cancer specimens by mass spectrometry confirm translation of the protein isoform. The P5P6 isoform is found to be constitutively phosphorylated, present in the cytoplasm, and it traffics to the plasma membrane. Expression of P5P6 in immortalized human pancreatic duct epithelial (HPDE) cells activates downstream AKT, and in human pancreatic epithelial nestin-expressing cells, activates both the AKT and MAPK pathways. Inhibiting RON P5P6 in HPDE cells using a small molecule inhibitor BMS-777607 blocked constitutive activation and decreased AKT signaling. P5P6 transforms NIH3T3 cells and induces tumorigenicity in HPDE cells. Resultant HPDE-P5P6 tumors develop a dense stromal compartment similar to that seen in pancreatic cancer. In summary, we have identified a novel and constitutively active isoform of the RON tyrosine kinase receptor that has transforming activity and is expressed in human pancreatic cancer. These findings provide additional insight into the biology of the RON receptor in pancreatic cancer and are clinically relevant to the study of RON as a potential therapeutic target. PMID:26477314

  4. Studying isoform-specific inositol 1,4,5-trisphosphate receptor function and regulation

    PubMed Central

    Betzenhauser, Matthew J.; Wagner, Larry E.; Won, Jong-Hak; Yule, David I.

    2011-01-01

    Inositol 1,4,5-trisphosphate receptors (InsP3R) are a family of ubiquitously expressed intracellular Ca2+ channels. Isoform-specific properties of the three family members may play a prominent role in defining the rich diversity of the spatial and temporal characteristics of intracellular Ca2+ signals. Studying the properties of the particular family members is complicated because individual receptor isoforms are typically never expressed in isolation. In this article, we discuss strategies for studying Ca2+ release through individual InsP3R family members with particular reference to methods applicable following expression of recombinant InsP3R and mutant constructs in the DT40-3KO cell line, an unambiguously null InsP3R expression system. PMID:18929664

  5. Reduced aerobic capacity causes leaky ryanodine receptors that trigger arrhythmia in a rat strain artificially selected and bred for low aerobic running capacity

    PubMed Central

    Høydal, MA; Stølen, TO; Johnsen, AB; Alvez, M; Catalucci, D; Condorelli, G; Koch, LG; Britton, SL; Smith, GL; Wisløff, U

    2014-01-01

    Aim Rats selectively bred for inborn Low Capacity of Running (LCR) display a series of poor health indices where as rats selected for High Capacity of Running (HCR) display a healthy profile. We hypothesized that selection of low aerobic capacity over generations leads to a phenotype with increased diastolic Ca2+ leak that trigger arrhythmia. Methods We used rats selected for HCR (N=10) or LCR (N=10) to determine the effect of inborn aerobic capacity on Ca2+ leak and susceptibility of ventricular arrhythmia. We studied isolated FURA2/AM loaded cardiomyocytes to detect Ca2+-handling and function on an inverted epi-fluorescence microscope. To determine arrhythmogenicity we did a final experiment with electrical burst pacing in Langendorff perfused hearts. Results Ca2+-handling was impaired by reduced Ca2+ amplitude, prolonged time to 50% Ca2+ decay, and reduced sarcoplasmic reticulum (SR) Ca2+-content. Impaired Ca2+ removal was influenced by reduced SR Ca2+ ATP-ase 2a (SERCA2a) function and increased sodium/Ca2+-exchanger (NCX) in LCR rats. Diastolic Ca2 leak was 87% higher in LCR rats. The leak was reduced by CaMKII inhibition. Expression levels of phosphorylated theorine-286 CaMKII levels and increased RyR2 phosphorylation at the Serine-2814 site mechanistically support our findings of increased leak in LCR. LCR rats had significantly higher incidence of ventricular fibrillation. Conclusion Selection of inborn low aerobic capacity over generations leads to a phenotype with increased risk of ventricular fibrillation. Increased phosphorylation of CaMKII at serine-2814 at the cardiac ryanodine receptor appears as an important mechanism of impaired Ca2+ handling and diastolic Ca2+ leak that results in increased susceptibility to ventricular fibrillation. PMID:24444142

  6. Differential neuroprotective and anti-inflammatory effects of L-type voltage dependent calcium channel and ryanodine receptor antagonists in the substantia nigra and locus coeruleus.

    PubMed

    Hopp, Sarah C; Royer, Sarah E; D'Angelo, Heather M; Kaercher, Roxanne M; Fisher, David A; Wenk, Gary L

    2015-03-01

    Neuroinflammation and degeneration of catecholaminergic brainstem nuclei occur early in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Neuroinflammation increases levels of pro-inflammatory cytokines and reactive oxygen species which can alter neuronal calcium (Ca(+2)) homoeostasis via L-type voltage dependent calcium channels (L-VDCCs) and ryanodine receptors (RyRs). Alterations in Ca(+2) channel activity in the SN and LC can lead to disruption of normal pacemaking activity in these areas, contributing to behavioral deficits. Here, we utilized an in vivo model of chronic neuroinflammation: rats were infused intraventricularly with a continuous small dose (0.25 μg/h) of lipopolysaccharide (LPS) or artificial cerebrospinal fluid (aCSF) for 28 days. Rats were treated with either the L-VDCC antagonist nimodipine or the RyR antagonist dantrolene. LPS-infused rats had significant motor deficits in the accelerating rotarod task as well as abnormal behavioral agitation in the forced swim task and open field. Corresponding with these behavioral deficits, LPS-infused rats also had significant increases in microglia activation and loss of tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra pars compacta (SNpc) and locus coeruleus (LC). Treatment with nimodipine or dantrolene normalized LPS-induced abnormalities in the rotarod and forced swim, restored the number of TH-immunoreactive cells in the LC, and significantly reduced microglia activation in the SNpc. Only nimodipine significantly reduced microglia activation in the LC, and neither drug increased TH immunoreactivity in the SNpc. These findings demonstrate that the Ca(+2) dysregulation in the LC and SN brainstem nuclei is differentially altered by chronic neuroinflammation. Overall, targeting Ca + 2 dysregulation may be an important target for ameliorating neurodegeneration in the SNpc and LC. PMID:25318607

  7. The effects of ryanodine receptor (RYR1) mutation on natural killer cell cytotoxicity, plasma cytokines and stress hormones during acute intermittent exercise in pigs.

    PubMed

    Ciepielewski, Z M; Stojek, W; Borman, A; Myślińska, D; Pałczyńska, P; Kamyczek, M

    2016-04-01

    Stress susceptibility has been mapped to a single recessive gene, the ryanodine receptor 1 (RYR1) gene or halothane (Hal) gene. Homozygous (Hal(nn)), mutated pigs are sensitive to halothane and susceptible to Porcine Stress Syndrome (PSS). Previous studies have shown that stress-susceptible RYR1 gene mutated homozygotes in response to restraint stress showed an increase in natural killer cell cytotoxicity (NKCC) accompanied by more pronounced stress-related hormone and anti-inflammatory cytokine changes. In order to determine the relationship of a RYR1 gene mutation with NKCC, plasma cytokines and stress-related hormones following a different stress model - exercise - 36 male pigs (representing different genotypes according to RYR1 gene mutation: NN, homozygous dominant; Nn, heterozygous; nn, homozygous recessive) were submitted to an intermittent treadmill walking. During the entire experiment the greatest level of NKCC and the greatest concentrations of interleukin (IL-) 6, IL-10, IL-12, interferon (IFN-)γ and tumor necrosis factor-α and stress-related hormones (adrenaline, prolactin, beta-endorphin) were observed in nn pigs, and the greatest concentration of IL-1 and growth hormone in NN pigs. Immunostimulatory effects of intermittent exercise on NKCC in nn pigs were concomitant with increases in IL-2, IL-12 and IFN-γ, the potent NKCC activators. Our findings suggest that stress-susceptible pigs RYR1 gene mutated pigs develop a greater level of NKCC and cytokine production in response to exercise stress. These results suggest that the heterogeneity of immunological and neuroendocrine response to exercise stress in pigs could be influenced by RYR1 gene mutation. PMID:27033913

  8. Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes

    PubMed Central

    Savio-Galimberti, Eleonora; Knollmann, Björn C.

    2015-01-01

    Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT. PMID:26121139

  9. Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes.

    PubMed

    Savio-Galimberti, Eleonora; Knollmann, Björn C

    2015-01-01

    Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT. PMID:26121139

  10. Contribution of impaired myofibril and ryanodine receptor function to prolonged low-frequency force depression after in situ stimulation in rat skeletal muscle.

    PubMed

    Watanabe, Daiki; Kanzaki, Keita; Kuratani, Mai; Matsunaga, Satoshi; Yanaka, Noriyuki; Wada, Masanobu

    2015-06-01

    The aim of this study was to examine whether prolonged low-frequency force depression (PLFFD) that occurs in situ is the result of decreased myofibrillar Ca(2+) sensitivity and/or reduced sarcoplasmic reticulum (SR) Ca(2+) release. Intact rat gastrocnemius muscles were electrically stimulated via the sciatic nerve until force was reduced to ~50% of the initial and dissected 30 min following the cessation of stimulation. Skinned fibre and whole muscle analyses were performed in the superficial region composed exclusively of type IIB fibres. Fatiguing stimulation significantly reduced the ratio of force at low frequency to that at high frequency to 65% in skinned fibres (1 vs. 50 Hz) and 73% in whole muscles (20 vs. 100 Hz). In order to evaluate changes in myofibrillar Ca(2+) sensitivity and ryanodine receptor caffeine sensitivity, skinned fibres were activated in Ca(2+)- and caffeine-containing solutions, respectively. Skinned fibres from fatigued muscles displayed decreased caffeine sensitivity together with increased myofibrillar Ca(2+) sensitivity. Treatment with 2,2'-dithiodipyridine and reduced glutathione induced a smaller increase in myofibrillar Ca(2+)sensitivity in fatigued than in rested fibres. In fatigued muscles, S-glutathionylation of troponin I was increased and submaximal SR Ca(2+) release, induced by 4-chloro-m-cresol, was decreased. These findings suggest that in the early stage of PLFFD that occurs in fast-twitch muscles of exercising animals and humans, S-glutathionylation of troponin I may attenuate PLFFD by increasing myofibrillar Ca(2+) sensitivity and that under such a circumstance, PLFFD may be ascribable to failure of SR Ca(2+) release. PMID:25697123

  11. Identification of Novel Ryanodine Receptor 1 (RyR1) Protein Interaction with Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)*♦

    PubMed Central

    Ryan, Timothy; Sharma, Parveen; Ignatchenko, Alex; MacLennan, David H.; Kislinger, Thomas; Gramolini, Anthony O.

    2011-01-01

    The ryanodine receptor type 1 (RyR1) is a homotetrameric Ca2+ release channel located in the sarcoplasmic reticulum of skeletal muscle where it plays a role in the initiation of skeletal muscle contraction. A soluble, 6×-histidine affinity-tagged cytosolic fragment of RyR1 (amino acids 1–4243) was expressed in HEK-293 cells, and metal affinity chromatography under native conditions was used to purify the peptide together with interacting proteins. When analyzed by gel-free liquid chromatography mass spectrometry (LC-MS), 703 proteins were identified under all conditions. This group of proteins was filtered to identify putative RyR interacting proteins by removing those proteins found in only 1 RyR purification and proteins for which average spectral counts were enriched by less than 4-fold over control values. This resulted in 49 potential RyR1 interacting proteins, and 4 were selected for additional interaction studies: calcium homeostasis endoplasmic reticulum protein (CHERP), endoplasmic reticulum-Golgi intermediate compartment 53-kDa protein (LMAN1), T-complex protein, and phosphorylase kinase. Western blotting showed that only CHERP co-purified with affinity-tagged RyR1 and was eluted with imidazole. Immunofluorescence showed that endogenous CHERP co-localizes with endogenous RyR1 in the sarcoplasmic reticulum of rat soleus muscle. A combination of overexpression of RyR1 in HEK-293 cells with siRNA-mediated suppression of CHERP showed that CHERP affects Ca2+ release from the ER via RyR1. Thus, we propose that CHERP is an RyR1 interacting protein that may be involved in the regulation of excitation-contraction coupling. PMID:21454501

  12. Synchrony of Cardiomyocyte Ca2+ Release is Controlled by t-tubule Organization, SR Ca2+ Content, and Ryanodine Receptor Ca2+ Sensitivity

    PubMed Central

    Øyehaug, Leiv; Loose, Kristian Ø.; Jølle, Guro F.; Røe, Åsmund T.; Sjaastad, Ivar; Christensen, Geir; Sejersted, Ole M.; Louch, William E.

    2013-01-01

    Recent work has demonstrated that cardiomyocyte Ca2+release is desynchronized in several pathological conditions. Loss of Ca2+ release synchrony has been attributed to t-tubule disruption, but it is unknown if other factors also contribute. We investigated this issue in normal and failing myocytes by integrating experimental data with a mathematical model describing spatiotemporal dynamics of Ca2+ in the cytosol and sarcoplasmic reticulum (SR). Heart failure development in postinfarction mice was associated with progressive t-tubule disorganization, as quantified by fast-Fourier transforms. Data from fast-Fourier transforms were then incorporated in the model as a dyadic organization index, reflecting the proportion of ryanodine receptors located in dyads. With decreasing dyadic-organization index, the model predicted greater dyssynchrony of Ca2+ release, which exceeded that observed in experimental line-scan images. Model and experiment were reconciled by reducing the threshold for Ca2+ release in the model, suggesting that increased RyR sensitivity partially offsets the desynchronizing effects of t-tubule disruption in heart failure. Reducing the magnitude of SR Ca2+ content and release, whether experimentally by thapsigargin treatment, or in the model, desynchronized the Ca2+ transient. However, in cardiomyocytes isolated from SERCA2 knockout mice, RyR sensitization offset such effects. A similar interplay between RyR sensitivity and SR content was observed during treatment of myocytes with low-dose caffeine. Initial synchronization of Ca2+ release during caffeine was reversed as SR content declined due to enhanced RyR leak. Thus, synchrony of cardiomyocyte Ca2+ release is not only determined by t-tubule organization but also by the interplay between RyR sensitivity and SR Ca2+ content. PMID:23601316

  13. Topographies and isoforms of the progesterone receptor in female human, rat and mouse bladder.

    PubMed

    Gevaert, Thomas; Rietjens, Roma; Voets, Thomas; Everaerts, Wouter; De Ridder, Dirk

    2016-05-01

    Steroid hormones such as progesterone are known to influence bladder function. Progesterone effects are mediated by the progesterone receptor (PR) but no detailed studies of PR in bladder exist. We have investigated the presence, topography and subtypes of PR in mouse, rat and human bladder. Fresh tissue samples were obtained from cystectomies in female humans, rats and mice (n = 7 per group). Tissue samples were processed for immunohistochemistry (IHC), immunofluorescence (IF) and western blot (WB) and, for each species, a panel of specific PR antibody clones was used. Interpretation of IHC/IF was carried out by light/fluorescent microscopy and of WB via standard WB software. IHC/IF in female human bladder showed PR on the interstitial cells in the lamina propria and between detrusor smooth muscle cells, whereas in female rat and mouse bladder, PR was only found on the urothelium. WB in human bladder showed a 78-kD and a 60-kDa band, respectively, corresponding to a modified PR isoform A and PR isoform C. WB in rat and mice bladder showed a 60 kDa band and a 37 kDa band, respectively corresponding with PR isoform C and an unknown isoform. This is the first detailed investigation of the precise location and presence of several isoforms of PR in bladder, together with a comparison of these data between human, rat and mouse. Our study has revealed complex PR families in bladders from the various species studied and demonstrates obvious inter-species differences in PR topography and isoforms. PMID:26650465

  14. Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

    PubMed Central

    Natali, P. G.; Nicotra, M. R.; Di Filippo, F.; Bigotti, A.

    1995-01-01

    In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo. Images Figure 1 PMID:7779718

  15. The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles.

    PubMed

    Diao, Feici; Mena, Wilson; Shi, Jonathan; Park, Dongkook; Diao, Fengqiu; Taghert, Paul; Ewer, John; White, Benjamin H

    2016-01-01

    To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced "Trojan exon" technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks. PMID:26534952

  16. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity

    PubMed Central

    Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W.

    2016-01-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies. PMID:26909609

  17. Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro

    PubMed Central

    Howarth, Deanna L.; Hagey, Lee R.; Law, Sheran H.W.; Ai, Ni; Krasowski, Matthew D.; Ekins, Sean; Moore, John T.; Kollitz, Erin M.; Hinton, David E.; Kullman, Seth W.

    2010-01-01

    The nuclear receptor farnesoid X receptor alpha (FXRα, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxrα in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXRα (~70% in the ligand-binding domain). Fxrα1 and Fxrα2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxrα1 having an extended N-terminus compared to Fxrα2. A Gal4DBD-FxrαLBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-FxrαLBD fusion construct was enhanced by addition of PGC-1α, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxrα isoforms were compared in transient transfection assays, Fxrα2 was activated by C24 bile acids and GW4064, while Fxrα1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AF1 domain, these experiments highlight a key functional region in the Fxrα AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxrα2, but not Fxrα1, to interact with PGC-1α and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxrα2 isoform) are activated by primary and secondary bile acids. PMID:20430454

  18. K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

    PubMed

    Darcy, Yuanzhao L; Diaz-Sylvester, Paula L; Copello, Julio A

    2016-08-01

    K201 (JTV-519) may prevent abnormal Ca(2+) leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca(2+)-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca(2+) loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca(2+)-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca(2+) and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca(2+) release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca(2+) release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active ("high-activity") RyR1 of SkM reconstituted into bilayers, but it had no effect on "low-activity" channels. Likewise, K201 activated cardiac RyR2 under systolic Ca(2+) conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca(2+) conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca(2+) leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca(2+) conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca(2+) overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca(2+) release. PMID:27235390

  19. Calcium-Dependent Energetics of Calmodulin Domain Interactions with Regulatory Regions of the Ryanodine Receptor Type 1 (RyR1)

    PubMed Central

    Newman, Rhonda A.; Sorensen, Brenda R.; Kilpatrick, Adina M.; Shea, Madeline A.

    2014-01-01

    Calmodulin (CaM) plays a vital role in calcium homeostasis by allosterically modulating intracellular calcium channels including the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1). Apo (calcium-free) CaM activates hRyR1 while calcium-saturated CaM inhibits it. Two CaM-binding regions (residues 1975–1999 and 3614–3643) identified in each RyR1 monomer were proposed to allow CaM to bridge adjacent RyR1 subunits. We explored the distinct roles of CaM domains by using fluorescence anisotropy to determine the affinity of CaM1–148 (full-length), CaM1–80 (N-domain) and CaM76–148 (C-domain) for peptides encompassing hRyR1 residues 1975–1999 or 3614–3643. Both CaM1–148 and CaM76–148 associated in a calcium-independent manner with similar affinities for hRyR1(3614–3643)p while CaM1–80 required calcium and bound ~250-fold more weakly. Association of CaM1–148, CaM1–80 and CaM76–148 with hRyR1(1975–1999)p was much less favorable than with hRyR1(3614–3643)p; differences between the two CaM domains were smaller. Equilibrium calcium titrations monitored by steady-state fluorescence demonstrated that both hRyR1 peptides increased the calcium-binding affinity of both CaM domains. These thermodynamic properties support a prior model in which the CaM C-domain associates with RyR1(3614–3643) at low levels of calcium, positioning CaM to rapidly respond to calcium efflux. However, the affinity of the N-domain of CaM for hRyR1(1975–1999)p is insufficient to explain a model in which CaM bridges adjacent RyR1 subunits within the tetramer. This indicates that other protein factors or properties of the tertiary or quaternary structure of hRyR1 contribute to the energetics of CaM-mediated regulation. PMID:25145833

  20. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor

    PubMed Central

    Eltit, Jose Miguel; Bannister, Roger A.; Moua, Ong; Altamirano, Francisco; Hopkins, Philip M.; Pessah, Isaac N.; Molinski, Tadeusz F.; López, Jose R.; Beam, Kurt G.; Allen, Paul D.

    2012-01-01

    Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca2+ release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation–contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and CaV1.1, the principal subunit of the L-type Ca2+ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca2+ entry. The CaV1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all CaV channels [Carpenter D, et al. (2009) BMC Med Genet 10:104–115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (CaV1.1 null) myotubes. Unlike previously described MH-linked mutations in CaV1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca2+ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in CaV1.1 R174W-expressing myotubes, resting myoplasmic Ca2+ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type CaV1.1. Our results indicate that CaV1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca2+ leak from the SR, and that perturbation of CaV1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers. PMID:22547813

  1. Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

    PubMed

    Eltit, Jose Miguel; Bannister, Roger A; Moua, Ong; Altamirano, Francisco; Hopkins, Philip M; Pessah, Isaac N; Molinski, Tadeusz F; López, Jose R; Beam, Kurt G; Allen, Paul D

    2012-05-15

    Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers. PMID:22547813

  2. Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility.

    PubMed

    Prestle, J; Janssen, P M; Janssen, A P; Zeitz, O; Lehnart, S E; Bruce, L; Smith, G L; Hasenfuss, G

    2001-02-01

    The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase-polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP-infected control cells (4.8+/-0.2% FS versus 4+/-0.2% FS, respectively; n=79 each; P:=0.001). SR-Ca(2+) uptake rates were monitored in beta-escin-permeabilized myocytes using Fura-2. Ad-FKBP12.6-infected cells showed a statistically significant higher rate of Ca(2+) uptake of 0.8+/-0.09 nmol/s(-)(1)/10(6) cells (n=8, P:<0.05) compared with 0.52+/-0.1 nmol/s(-)(1)/10(6) cells in sham-infected cells (n=8) at a [Ca(2+)] of 1 micromol/L. In the presence of 5 micromol/L ruthenium red to block Ca(2+) efflux via RyR2, SR-Ca(2+) uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca(2+) leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6-infected myocytes compared with Ad-GFP-infected control cells, indicating a higher SR-Ca(2+) load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca(2+) leak and consequently increase SR-Ca(2+) release and myocyte shortening. PMID:11157671

  3. Redox modification of ryanodine receptors by mitochondria-derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts.

    PubMed

    Cooper, Leroy L; Li, Weiyan; Lu, Yichun; Centracchio, Jason; Terentyeva, Radmila; Koren, Gideon; Terentyev, Dmitry

    2013-12-01

    Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca(2+)) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca(2+) handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5-9 months) and old (4-6 years) rabbit hearts to test the hypothesis that changes in Ca(2+) homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca(2+) handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca(2+) dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca(2+) load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca(2+) leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca(2+) transient amplitude, SR Ca(2+) load, and latency of pro-arrhythmic spontaneous Ca(2+) waves (SCWs) were decreased while RyR-mediated SR Ca(2+) leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca(2+) leak, ablated most

  4. Rank-based genome-wide analysis reveals the association of Ryanodine receptor-2 gene variants with childhood asthma among human populations

    PubMed Central

    2013-01-01

    Background The standard approach to determine unique or shared genetic factors across populations is to identify risk alleles in one population and investigate replication in others. However, since populations differ in DNA sequence information, allele frequencies, effect sizes, and linkage disequilibrium patterns, SNP association using a uniform stringent threshold on p values may not be reproducible across populations. Here, we developed rank-based methods to investigate shared or population-specific loci and pathways for childhood asthma across individuals of diverse ancestry. We performed genome-wide association studies on 859,790 SNPs genotyped in 527 affected offspring trios of European, African, and Hispanic ancestry using publically available asthma database in the Genotypes and Phenotypes database. Results Rank-based analyses showed that there are shared genetic factors for asthma across populations, more at the gene and pathway levels than at the SNP level. Although the top 1,000 SNPs were not shared, 11 genes (RYR2, PDE4D, CSMD1, CDH13, ROBO2, RBFOX1, PTPRD, NPAS3, PDE1C, SEMA5A, and CTNNA2) mapped by these SNPs were shared across populations. Ryanodine receptor 2 (RYR2, a statin response-related gene) showed the strongest association in European (p value = 2.55 × 10−7) and was replicated in African (2.57 × 10−4) and Hispanic (1.18 × 10−3) Americans. Imputation analyses based on the 1000 Genomes Project uncovered additional RYR2 variants associated with asthma. Network and functional ontology analyses revealed that RYR2 is an integral part of dermatological or allergic disorder biological networks, specifically in the functional classes involving inflammatory, eosinophilic, and respiratory diseases. Conclusion Our rank-based genome-wide analysis revealed for the first time an association of RYR2 variants with asthma and replicated previously discovered PDE4D asthma gene across human populations. The replication of top

  5. Increased Ca2+ sensitivity of the ryanodine receptor mutant RyR2R4496C underlies catecholaminergic polymorphic ventricular tachycardia.

    PubMed

    Fernández-Velasco, María; Rueda, Angélica; Rizzi, Nicoletta; Benitah, Jean-Pierre; Colombi, Barbara; Napolitano, Carlo; Priori, Silvia G; Richard, Sylvain; Gómez, Ana María

    2009-01-30

    Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca(2+) handling underlie this disease. Here we analyze the underlying Ca(2+) release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2(R4496C) mutation. RyR2(R4496C-/-) littermates (wild type) were used as controls. [Ca(2+)](i) transients were obtained by field stimulation in fluo-3-loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca(2+)](i) transients and sarcoplasmic reticulum Ca(2+) load were similar in wild-type and RyR2(R4496C) cells. However, paced RyR2(R4496C) ventricular myocytes presented abnormal Ca(2+) release during the diastolic period, viewed as Ca(2+) waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca(2+) release was enhanced at faster stimulation rates and by beta-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca(2+) sparks were more frequent in RyR2(R4496C) myocytes, indicating increased RyR2(R4496C) activity. When permeabilized cells were exposed to different cytosolic [Ca(2+)](i), RyR2(R4496C) showed a dramatic increase in Ca(2+) sensitivity. Isoproterenol increased [Ca(2+)](i) transient amplitude and Ca(2+) spark frequency to the same extent in wild-type and RyR2(R4496C) cells, indicating that the beta-adrenergic sensitivity of RyR2(R4496C) cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2(R4496C) mutation is attributable to the increased Ca(2+) sensitivity of RyR2(R4496C), which induces diastolic Ca(2+) release and lowers the threshold for triggered activity. PMID:19096022

  6. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

    PubMed

    Cao, Yun; Bender, Ingrid K; Konstantinidis, Athanasios K; Shin, Soon Cheon; Jewell, Christine M; Cidlowski, John A; Schleimer, Robert P; Lu, Nick Z

    2013-02-28

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  7. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans

    PubMed Central

    Cao, Yun; Bender, Ingrid K.; Konstantinidis, Athanasios K.; Shin, Soon Cheon; Jewell, Christine M.; Cidlowski, John A.; Schleimer, Robert P.

    2013-01-01

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  8. Alternative splicing in the fiddler crab cognate ecdysteroid receptor: variation in receptor isoform expression and DNA binding properties in response to hormone.

    PubMed

    Durica, David S; Das, Sunetra; Najar, Fares; Roe, Bruce; Phillips, Barret; Kappalli, Sudha; Anilkumar, Gopinathan

    2014-09-15

    RXR cDNA cloning from three Uca species led to the identification of 4 conserved isoforms, indicative of alternative splicing in the hinge and ligand binding domains (LBD). Sequencing of overlapping clones from a Ucapugilator genomic library identified EcR isoforms matching previously identified cDNA variants; in addition, a cryptic exon in the LBD was detected and evidence for expression of this new isoform was obtained from next-generation sequencing. RNA-seq analysis also identified a new amino terminal EcR variant. EcR and RXR transcript abundance increases throughout ovarian maturation in U. pugilator, while cognate receptor transcript abundance remains constant in a related Indo-Pacific species with a different reproductive strategy. To examine if crab RXR LBD isoforms have different physical properties in vitro, electromobility shift assays were performed with different EcR isoforms. The cognate crab and fruit fly receptors differ in their responses to hormone. Ecdysteroids did not increase DNA binding for the crab heterodimers, while ecdysteroids stimulate binding for Drosophilamelanogaster EcR/USP heterodimers. In swapping experiments, UpEcR/USP heterodimers did not show ligand-responsive differences in DNA binding; both crab RXR LBD isoforms, however, conferred ligand-responsive increases in DNA binding with DmEcRs. These data indicate that both UpRXR LBD isoforms can heterodimerize with the heterologous DmEcR receptors and promote ligand and DNA binding. Unresponsiveness of the cognate receptors to ecdysteroid, however, suggest additional factors may be required to mediate endogenous, perhaps isoform-specific, differences in EcR conformation, consistent with previously reported effects of UpRXR isoforms on UpEcR ligand-binding affinities. PMID:25025945

  9. Steroid Receptor Isoform Expression in Drosophila Nociceptor Neurons Is Required for Normal Dendritic Arbor and Sensitivity

    PubMed Central

    McParland, Aidan L.; Follansbee, Taylor L.; Vesenka, Gwendolyn D.; Panaitiu, Alexandra E.; Ganter, Geoffrey K.

    2015-01-01

    Steroid hormones organize many aspects of development, including that of the nervous system. Steroids also play neuromodulatory and other activational roles, including regulation of sensitivity to painful stimuli in mammals. In Drosophila, ecdysteroids are the only steroid hormones, and therefore the fly represents a simplified model system in which to explore mechanisms of steroid neuromodulation of nociception. In this report, we present evidence that ecdysteroids, acting through two isoforms of their nuclear ecdysone receptor (EcR), modulate sensitivity to noxious thermal and mechanical stimuli in the fly larva. We show that EcRA and EcRB1 are expressed by third instar larvae in the primary nociceptor neurons, known as the class IV multidendritic neurons. Suppression of EcRA by RNA interference in these cells leads to hyposensitivity to noxious stimulation. Suppression of EcRB1 leads to reduction of dendritic branching and length of nociceptor neurons. We show that specific isoforms of the ecdysone receptor play critical cell autonomous roles in modulating the sensitivity of nociceptor neurons and may indicate human orthologs that represent targets for novel analgesic drugs. PMID:26495837

  10. Altered expression of progesterone receptor isoforms A and B in human eutopic endometrium in endometriosis patients.

    PubMed

    Wölfler, Monika Martina; Küppers, Mareike; Rath, Werner; Buck, Volker Uwe; Meinhold-Heerlein, Ivo; Classen-Linke, Irmgard

    2016-07-01

    Recent data implicate an altered expression of progesterone receptor isoform A (PR-A) and B (PR-B) in the endometrium of endometriosis patients. This prospective exploratory study aimed to precisely determine the PR-A and PR-B expression using immunohistochemical techniques in eutopic endometrium of women with endometriosis compared with disease-free women throughout the menstrual cycle. All symptomatic patients underwent laparoscopy for the diagnosis of endometriosis and histological confirmation of the disease (EO) whereas controls were proven disease-free (CO). In CO samples (n=10) an increased expression of PR-A and PR-B during the proliferative to early secretory phase and a decreased expression of both receptor isoforms during the mid to late secretory phase was ascertained in accordance with previous studies. In patients with endometriosis (n=16) no cycle dependent pattern of PR-A and PR-B expression was identified in contrast to patients without endometriosis. Moreover, in EO samples a huge variety of inter- and intra-individual differences in PR-A and PR-B expression were detected. These data provide further evidence that dysregulation of the PR-A and PR-B expression might contribute to the pathophysiology of endometriosis. PMID:27050108

  11. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging

    PubMed Central

    2014-01-01

    Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments. PMID:24935247

  12. Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform.

    PubMed

    Basu, Dipannita; Castellano, Jessica M; Thomas, Nancy; Mishra, Ram K

    2013-01-01

    The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100/D-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. PMID:23424095

  13. [Isoforms of the human histamine H3 receptor: Generation, expression in the central nervous system and functional implications].

    PubMed

    García-Gálvez, Ana Maricela; Arias-Montaño, José Antonio

    2016-01-01

    Histamine plays a significant role as a neuromodulator in the human central nervous system. Histamine-releasing neurons are exclusively located in the tuberomammillary nucleus of the hypothalamus, project to all major areas of the brain, and participate in functions such as the regulation of sleep/wakefulness, locomotor activity, feeding and drinking, analgesia, learning, and memory. The functional effects of histamine are exerted through the activation of four G protein-coupled receptors (H1, H2, H3 and H4), and in the central nervous system the first three receptors are widely expressed. The H3 receptor (H3R) is found exclusively in neuronal cells, where it functions as auto- and hetero-receptor. One remarkable characteristic of the H3R is the existence of isoforms, generated by alternative splicing of the messenger RNA. For the human H3R, 20 isoforms have been reported; although a significant number lack those regions required for agonist binding or receptor signaling, at least five isoforms appear functional upon heterologous expression. In this work we review the evidence for the generation of human H3R isoforms, their expression, and the available information regarding the functionality of such receptors. PMID:26927649

  14. Purification and characterization of ryanotoxin, a peptide with actions similar to those of ryanodine.

    PubMed Central

    Morrissette, J; Beurg, M; Sukhareva, M; Coronado, R

    1996-01-01

    We purified and characterized ryanotoxin, an approximately 11.4-kDa peptide from the venom of the scorpion Buthotus judiacus that induces changes in ryanodine receptors of rabbit skeletal muscle sarcoplasmic reticulum analogous to those induced by the alkaloid ryanodine. Ryanotoxin stimulated Ca2+ release from sarcoplasmic reticulum vesicles and induced a state of reduce unit conductance with a mean duration longer than that of unmodified ryanodine receptor channels. With Cs+ as the current carrier, the slope conductance of the state induced by 1 microM ryanotoxin was 163 +/- 12 pS, that of the state induced by 1 microM ryanodine was 173 +/- 26 pS, and that of control channels was 2.3-fold larger (396 +/- 25 pS). The distribution of substate events induced by 1 microM RyTx was biexponential and was fitted with time constants approximately 10 times shorter than those fitted to the distribution of substates induced by 1 microM ryanodine. Bath-applied 5 microM ryanotoxin had no effect on the excitability of mouse myotubes in culture. When 5 microM ryanotoxin was dialyzed into the cell through the patch pipette in the whole-cell configuration, there was a voltage-dependent increase in the amplitude of intracellular Ca2+ transients elicited by depolarizing potentials in the range of -30 to +50 mV. Ryanotoxin increased the binding affinity of [3H]ryanodine in a reversible manner with a 50% effective dose (ED50) of 0.16 microM without altering the maximum number (Bmax) of [3H]ryanodine-binding sites. This result suggested that binding sites for ryanotoxin and ryanodine were different. Ryanotoxin should prove useful in identifying domains coupling the ryanodine receptor to the voltage sensor, or domains affecting the gating and conductance of the ryanodine receptor channel. Images FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 11 PMID:8842209

  15. Identification of novel chicken estrogen receptor-alpha messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage.

    PubMed

    Griffin, C; Flouriot, G; Sonntag-Buck, V; Nestor, P; Gannon, F

    1998-11-01

    Using the rapid amplification of complementary DNA ends (RACE) methodology we have identified three new chicken estrogen receptor-alpha (cER alpha) messenger RNA (mRNA) variants in addition to the previously described form (isoform A). Whereas one of the new variants (isoform B) presents a 5'-extremity contiguous to the 5'-end of isoform A, the two other forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1 (A). The 3'-end of exon 1C has been located at position -1334 upstream of the transcription start site of the A isoform (+1). Whereas the genomic location of exon 1D is unknown, 700 bp 5' to this exon were isolated by genomic walking, and their sequence was determined. The transcription start sites of the cER alpha mRNA isoforms were defined. In transfection experiments, the regions immediately upstream of the A-D cER alpha mRNA isoforms were shown to possess cell-specific promoter activities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that the expression of the four different cER alpha mRNA isoforms is under the control of four different promoters. Finally, RT-PCR, S1 nuclease mapping, and primer extension analysis of these different cER alpha mRNA isoforms revealed a differential pattern of expression of the cER alpha gene in chicken tissues. Together, the results suggest that alternative 5'-splicing and promoter usage may be mechanisms used to modulate the levels of expression of the chicken ER alpha gene in a tissue-specific and/or developmental stage-specific manner. PMID:9794473

  16. Aberrant Liver Insulin Receptor Isoform A Expression Normalises with Remission of Type 2 Diabetes after Gastric Bypass Surgery

    PubMed Central

    Besic, Vinko; Shi, Hongjun; Stubbs, Richard S.; Hayes, Mark T.

    2015-01-01

    Type 2 diabetes mellitus (T2DM) results from a combination of progressive insulin resistance and loss of pancreatic beta cell function and/or mass. Insulin signalling occurs through the insulin receptor, (INSR) which is alternatively spliced into two isoforms: INSRA (-exon 11) and INSRB (+exon 11). Because the INSR isoforms have different functional characteristics, their relative expression ratio has been implicated in the pathogenesis of insulin resistance and T2DM. We studied levels of INSR isoform mRNA in liver samples taken from 46 individuals with or without T2DM at Roux-en-Y (RYGB) surgery, and on average 17 (± 5.6) months later in 16 of the same individuals (8 diabetic and non-diabetic patients). INSRA or INSRB was also overexpressed in HepG2 cells to ascertain their effect on AKT phosphorylation and PCK1 expression as markers of insulin-mediated metabolic signalling. We found the INSRB:A isoform ratio was reduced in individuals with T2DM in comparison to those with normal glucose tolerance and normalised with remission of diabetes. The INSRB:A ratio increased due to a reduction in the alternatively spliced INSRA isoform following remission of diabetes. Overexpressing INSRA isoform in HepG2 hepatoma cells reduced inhibition of PCK1 transcription and did not increase AKT phosphorylation in response to insulin load compared to the effect of overexpressing the B isoform. Data presented here revitalizes the role of the INSR isoforms in the pathogenesis of T2DM, and suggests that an abrogated INSRB:A ratio that favours the INSRA isoform may negatively impact insulin-mediated metabolic signalling. PMID:25742416

  17. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  18. Differential function and regulation of orphan nuclear receptor TR3 isoforms in endothelial cells

    PubMed Central

    Zhou, Lei; Cui, Pengfei; Zhao, Shengqiang; Ye, Taiyang; Li, Yan; Peng, Jin; Niu, Gengming; Zhao, Dezheng

    2016-01-01

    TR3 has been reported to be an excellent target for angiogenesis therapies. We reported three TR3 transcript variant messenger RNAs (mRNAs) are expressed in human umbilical vein endothelial cell (HUVEC) and are differentially regulated by vascular endothelial growth factor (VEGF). TR3 transcript variant 1 (TR3-TV1) and variant 2 (TR3-TV2) encoding the same TR3 isoform 1 protein (TR3-iso1) that was named TR3 has been extensively studied. However, the function of TR3 isoform 2 protein (TR3-iso2) encoded by TR3 transcript variant 3 (TR3-TV3) is still not known. Here, we clone and express the novel TR3-iso2 protein and find that expression of TR3-iso2, in contrast to TR3-iso1, inhibits en-dothelial cell proliferation induced by VEGF-A, histamine, and phorbol-12-myristate-13-acetate (PMA). The differential function of TR3-iso2 correlates with the down-regulation of cyclin D1. However, TR3-iso2 plays similar roles in endothelial cell migration and monolayer permeability as TR3-iso1. We further demonstrate that several intracellular signaling pathways are involved in histamine-induced TR3 transcript variants, including histamine receptor H1-mediated phospholipase C (PLC)/calcium/calcineurin/protein kinase C (PKC)/ protein kinase D (PKD) pathway and ERK pathway, as well as histamine receptor H3-mediated PKC-ERK pathway. Further, expressions of TR3-TV1, TR3-TV2, and TR3-TV3 by VEGF and histamine are regulated by different promoters, but not by their mRNA stability. PMID:26440050

  19. INTERACTION OF PAH-RELATED COMPOUNDS WITH THE ALPHA AND BETA ISOFORMS OF ESTROGEN RECEPTOR. (R826192)

    EPA Science Inventory

    The ability of several 4- and 5-ring polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs, and their monohydroxy derivatives to interact with the estrogen receptor (ER) alpha and beta isoforms was examined. Only compounds possessing a hydroxyl group were able to compete wit...

  20. Epimorphin is a novel regulator of the progesterone receptor isoform-a.

    PubMed

    Bascom, Jamie L; Radisky, Derek C; Koh, Eileen; Fata, Jimmie E; Lo, Alvin; Mori, Hidetoshi; Roosta, Neda; Hirai, Yohei; Bissell, Mina J

    2013-09-15

    Epimorphin/syntaxin-2 is a membrane-tethered protein localized extracellularly (Epim) and intracellularly (Stx-2). The extracellular form Epim stimulates morphogenic processes in a range of tissues, including in murine mammary glands where its overexpression in luminal epithelial cells is sufficient to drive hyperplasia and neoplasia. We analyzed WAP-Epim transgenic mice to gain insight into how Epim promotes malignancy. Ectopic overexpression of Epim during postnatal mammary gland development led to early side-branching onset, precocious bud formation, and increased proliferation of mammary epithelial cells. Conversely, peptide-based inhibition of Epim function reduced side branching. Because increased side branching and hyperplasia occurs similarly in mice upon overexpression of the progesterone receptor isoform-a (Pgr-a), we investigated whether Epim exhibits these phenotypes through Pgr modulation. Epim overexpression indeed led to a steep upregulation of both total Pgr mRNA and Pgr-a protein levels. Notably, the Pgr antagonist RU486 abrogated Epim-induced ductal side branching, mammary epithelial cell proliferation, and bud formation. Evaluation of Epim signaling in a three-dimensional ex vivo culture system showed that its action was dependent on binding to its extracellular receptor, integrin-αV, and on matrix metalloproteinase 3 activity downstream of Pgr-a. These findings elucidate a hitherto unknown transcriptional regulator of Pgr-a, and shed light on how overexpression of Epim leads to malignancy. PMID:23867473

  1. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

    PubMed Central

    Chen, Nai-Yu; Nagarajan, Govindarajulu; Chiou, Pinwen Peter

    2015-01-01

    Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. PMID:25955250

  2. B cell receptor-mediated apoptosis of human lymphocytes is associated with a new regulatory pathway of Bim isoform expression.

    PubMed

    Mouhamad, Shahul; Besnault, Laurence; Auffredou, Marie Thérèse; Leprince, Corinne; Bourgeade, Marie Françoise; Leca, Gérald; Vazquez, Aimé

    2004-02-15

    Studies in Bim-deficient mice have shown that the proapoptotic molecule Bim plays a key role in the control of B cell homeostasis and activation. However, the role of Bim in human B lymphocyte apoptosis is unknown. We show in this study that, depending on the degree of cross-linking, B cell receptors can mediate both Bim-dependent and apparent Bim-independent apoptotic pathways. Cross-linked anti-mu Ab-mediated activation induces an original pathway governing the expression of the various Bim isoforms. This new pathway involves the following three sequential steps: 1) extracellular signal-regulated kinase-dependent phosphorylation of the BimEL isoform, which is produced in large amounts in healthy B cells; 2) proteasome-mediated degradation of phosphorylated BimEL; and 3) increased expression of the shorter apoptotic isoforms BimL and BimS. PMID:14764673

  3. Expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor in human alcoholic brain.

    PubMed

    Lewohl, J M; Crane, D I; Dodd, P R

    1997-03-14

    The expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the alpha class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because alpha 1 expression was raised in both groups of alcoholics of controls. The two groups of alcoholics could be differentiated on the basis of regional variations in alpha 1 expression. In frontal cortex, alpha 1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, alpha 1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform. PMID:9098573

  4. Cigarette Smoke and the Induction of Urokinase Plasminogen Activator Receptor In Vivo: Selective Contribution of Isoforms to Bronchial Epithelial Phenotype.

    PubMed

    Portelli, Michael A; Stewart, Ceri E; Hall, Ian P; Brightling, Christopher E; Sayers, Ian

    2015-08-01

    The urokinase plasminogen activator receptor (uPAR) gene (PLAUR) has been identified as an asthma susceptibility gene, with polymorphisms within that gene being associated with baseline lung function, lung function decline, and lung function in a smoking population. Soluble cleaved uPAR (scuPAR), a molecule identified as a marker of increased morbidity and mortality in a number of diseases, has been shown to be elevated in the airways of patients with asthma and in patients with chronic obstructive pulmonary disease. However, the functionality of soluble receptor isoforms and their relationship with an important initiator for obstructive lung disease, cigarette smoke, remains undefined. In this study, we set out to determine the effect of cigarette smoke on soluble uPAR isoforms, its regulatory pathway and the resultant effect on bronchial epithelial cell function. We identified a positive association between cigarette pack-years and uPAR expression in the airway bronchial epithelium of biopsies from patients with asthma (n = 27; P = 0.0485). In vitro, cigarette smoke promoted cleavage of uPAR from the surface of bronchial epithelial cells (1.5× induction; P < 0.0001) and induced the soluble spliced isoform through changes in messenger RNA expression (∼2× change; P < 0.001), driven by loss of endogenous 3' untranslated region suppression. Elevated expression of the soluble isoforms resulted in a proremodeling cell phenotype, characterized by increased proliferation and matrix metalloproteinase-9 expression in primary bronchial epithelial cells. This suggests that cigarette smoke elevates soluble receptor isoforms in bronchial epithelial cells through direct (cleavage) and indirect (messenger RNA expression) means. These findings provide further insight into how cigarette smoke may influence changes in the airways of importance to airway remodeling and obstructive lung disease progression. PMID:25490122

  5. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression

    PubMed Central

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required. PMID:26474308

  6. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

    PubMed

    Esber, Nathalie; Le Billan, Florian; Resche-Rigon, Michèle; Loosfelt, Hugues; Lombès, Marc; Chabbert-Buffet, Nathalie

    2015-01-01

    The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required. PMID:26474308

  7. Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

    PubMed

    Chan, Harvey Y S; Cheung, Man Chun; Gao, Yi; Miller, Andrew L; Webb, Sarah E

    2016-08-01

    In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation. PMID:27430888

  8. Estradiol differentially induces progesterone receptor isoforms expression through alternative promoter regulation in a mouse embryonic hypothalamic cell line.

    PubMed

    Vázquez-Martínez, Edgar Ricardo; Camacho-Arroyo, Ignacio; Zarain-Herzberg, Angel; Rodríguez, María Carmen; Mendoza-Garcés, Luciano; Ostrosky-Wegman, Patricia; Cerbón, Marco

    2016-06-01

    Progesterone receptor (PR) presents two main isoforms (PR-A and PR-B) that are regulated by two specific promoters and transcribed from alternative transcriptional start sites. The molecular regulation of PR isoforms expression in embryonic hypothalamus is poorly understood. The aim of the present study was to assess estradiol regulation of PR isoforms in a mouse embryonic hypothalamic cell line (mHypoE-N42), as well as the transcriptional status of their promoters. MHypoE-N42 cells were treated with estradiol for 6 and 12 h. Then, Western blot, real-time quantitative reverse transcription polymerase chain reaction, and chromatin and DNA immunoprecipitation experiments were performed. PR-B expression was transiently induced by estradiol after 6 h of treatment in an estrogen receptor alpha (ERα)-dependent manner. This induction was associated with an increase in ERα phosphorylation (serine 118) and its recruitment to PR-B promoter. After 12 h of estradiol exposure, a downregulation of this PR isoform was associated with a decrease of specific protein 1, histone 3 lysine 4 trimethylation, and RNA polymerase II occupancy on PR-B promoter, without changes in DNA methylation and hydroxymethylation. In contrast, there were no estradiol-dependent changes in PR-A expression that could be related with the epigenetic marks or the transcription factors evaluated. We demonstrate that PR isoforms are differentially regulated by estradiol and that the induction of PR-B expression is associated to specific transcription factors interactions and epigenetic changes in its promoter in embryonic hypothalamic cells. PMID:26676302

  9. Progesterone receptor isoforms PRA and PRB differentially contribute to breast cancer cell migration through interaction with focal adhesion kinase complexes

    PubMed Central

    Bellance, Catherine; Khan, Junaid A.; Meduri, Geri; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2013-01-01

    Progesterone receptor (PR) and progestins affect mammary tumorigenesis; however, the relative contributions of PR isoforms A and B (PRA and PRB, respectively) in cancer cell migration remains elusive. By using a bi-inducible MDA-MB-231 breast cancer cell line expressing PRA and/or PRB, we analyzed the effect of conditional PR isoform expression. Surprisingly, unliganded PRB but not PRA strongly enhanced cell migration as compared with PR(–) cells. 17,21-Dimethyl-19-norpregna-4,9-dien-3,20-dione (R5020) progestin limited this effect and was counteracted by the antagonist 11β-(4-dimethyl­amino)­phenyl-17β-hydroxy-17-(1-propynyl)­estra-4,9-dien-3-one (RU486). Of importance, PRA coexpression potentiated PRB-mediated migration, whereas PRA alone was ineffective. PR isoforms differentially regulated expressions of major players of cell migration, such as urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor type 1, uPA receptor (uPAR), and β1-integrin, which affect focal adhesion kinase (FAK) signaling. Moreover, unliganded PRB but not PRA enhanced FAK Tyr397 phosphorylation and colocalized with activated FAK in cell protrusions. Because PRB, as well as PRA, coimmunoprecipitated with FAK, both isoforms can interact with FAK complexes, depending on their respective nucleocytoplasmic trafficking. In addition, FAK degradation was coupled to R5020-dependent turnovers of PRA and PRB. Such an effect of PRB/PRA expression on FAK signaling might thus affect adhesion/motility, underscoring the implication of PR isoforms in breast cancer invasiveness and metastatic evolution with underlying therapeutic outcomes. PMID:23485561

  10. Positioning of the α-subunit isoforms confers a functional signature to γ-aminobutyric acid type A receptors

    PubMed Central

    Minier, Frédéric; Sigel, Erwin

    2004-01-01

    Fast synaptic inhibitory transmission in the CNS is mediated by γ-aminobutyric acid type A (GABAA) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different α-subunit isoforms, α1 and α6, in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized γ2β2α1β2α1, γ2β2α6β2α6, γ2β2α1β2α6, and γ2β2α6β2α1 GABAA receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single α6-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the α-subunit neighboring the γ2-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain α1- and α6-subunits. This method may also be applied to the study of other ion channels. PMID:15136735

  11. Thermodynamic analysis of progesterone receptor-promoter interactions reveals a molecular model for isoform-specific function.

    PubMed

    Connaghan-Jones, Keith D; Heneghan, Aaron F; Miura, Michael T; Bain, David L

    2007-02-13

    Human progesterone receptors (PR) exist as two functionally distinct isoforms, PR-A and PR-B. The proteins are identical except for an additional 164 residues located at the N terminus of PR-B. To determine the mechanisms responsible for isoform-specific functional differences, we present here a thermodynamic dissection of PR-A-promoter interactions and compare the results to our previous work on PR-B. This analysis has generated a number of results inconsistent with the traditional, biochemically based model of receptor function. Specifically, statistical models invoking preformed PR-A dimers as the active binding species demonstrate that intrinsic binding energetics are over an order of magnitude greater than is apparent. High-affinity binding is opposed, however, by a large energetic penalty. The consequences of this penalty are 2-fold: Successive monomer binding to a palindromic response element is thermodynamically favored over preformed dimer binding, and DNA-induced dimerization of the monomers is largely abolished. Furthermore, PR-A binding to multiple PREs is only weakly cooperative, as judged by a 5-fold increase in overall stability. Comparison of these results to our work on PR-B demonstrates that whereas both isoforms appear to have similar DNA binding affinities, PR-B in fact has a greatly increased intrinsic binding affinity and cooperative binding ability relative to PR-A. These differences thus suggest that residues unique to PR-B allosterically regulate the energetics of cooperative promoter assembly. From a functional perspective, the differences in microscopic affinities predict receptor-promoter occupancies that accurately correlate with the transcriptional activation profiles seen for each isoform. PMID:17277083

  12. Hypertrophy of lymphoid organs is a possible phenotypic characteristic of R420W mutation of the cardiac ryanodine receptor gene: a study using a knock-in mouse model.

    PubMed

    Nishio, Hajime; Okudaira, Noriyuki; Matsushita, Kazufumi; Yoshimoto, Tomohiro; Sato, Takako; Suzuki, Koichi

    2014-11-01

    Cardiac ryanodine receptor gene (RyR2) mutations sometimes result in sudden cardiac death due to fatal arrhythmias. N-terminal R420W mutation of RyR2 is known to show similar phenotypes to arrhythmogenic right ventricular cardiomyopathy and to cause juvenile sudden death. We previously reported two sudden death cases with the same R420W mutation. Interestingly, the cases showed hypertrophy of lymphoid organs such as the thymus and mesenteric lymph nodes. The present study examined whether R420W mutation of RYR2 causes hypertrophy of lymphoid organs by generating a mouse model carrying the mutation. Homozygous (RyR2(R420W/R420W)) mice showed significant increases in thymus and spleen weights but not in kidney, heart, and brain weights compared with wild-type mice. The mice also showed remarkable hypertrophy of mesenteric lymph nodes. Immunohistochemical study revealed that RyR2 protein was prominently expressed in epithelial cells of the thymic medulla in the thymus. These findings show that mice with R420W mutation of RyR2 exhibit hypertrophy of lymphoid organs. Sudden unexplained death cases with the mutation may display such findings at autopsy. PMID:25087098

  13. Immunohistochemical analysis of aldehyde dehydrogenase isoforms and their association with estrogen-receptor status and disease progression in breast cancer

    PubMed Central

    Opdenaker, Lynn M; Arnold, Kimberly M; Pohlig, Ryan T; Padmanabhan, Jayasree S; Flynn, Daniel C; Sims-Mourtada, Jennifer

    2014-01-01

    In many types of tumors, especially breast tumors, aldehyde dehydrogenase (ALDH) activity has been used to identify cancer stem-like cells within the tumor. The presence and quantity of these cells are believed to predict the response of tumors to chemotherapy. Therefore, identification and eradication of these cells would be necessary to cure the patient. However, there are 19 different ALDH isoforms that could contribute to the enzyme activity. ALDH1A1 and ALDH1A3 are among the isoforms mostly responsible for the increased ALDH activity observed in these stem-like cells, although the main isoforms vary in different tissues and tumor types. In the study reported here, we attempted to determine if ALDH1A1 or ALDH1A3, specifically, correlate with tumor stage, grade, and hormone-receptor status in breast-cancer patients. While there was no significant correlation between ALDH1A1 and any of the parameters tested, we were able to identify a positive correlation between ALDH1A3 and tumor stage in triple-negative cancers. In addition, ALDH1A3 was negatively correlated with estrogen-receptor status. Our data suggest that ALDH1A3 could be utilized as a marker to identify stem-like cells within triple-negative tumors. PMID:25540596

  14. Cellular localization and changes in expression of prolactin receptor isoforms in sheep ovary throughout the estrous cycle.

    PubMed

    Picazo, R A; García Ruiz, J P; Santiago Moreno, J; González de Bulnes, A; Muñoz, J; Silván, G; Lorenzo, P L; Illera, J C

    2004-11-01

    The actions of prolactin (PRL) on target cells depend on the type of prolactin receptor (PRLr) predominantly expressed, particularly whether the long PRLr isoform is expressed. The aims of this study were to determine the cellular localization and the changes in expression of long and short PRLr isoforms in sheep ovary throughout the estrous cycle. Long and short PRLrs were localized mostly in the same ovarian cells. Maximum signal intensity, particularly for long PRLrs, was found in stromal cells surrounding primordial and primary follicles, and, for both PRLrs, in granulosa cells of preantral follicles and in luteal cells. Moderate signal intensity for PRLrs was found in theca cells of preantral to ovulatory follicles, and in granulosa cells of antral follicles up to the gonadotropin-dependent stage. Decreasing immunoreactivity to PRLrs was found in granulosa cells of gonadotropin-dependent to ovulatory follicles. For long PRLrs in particular, no signal was found in mural granulosa cells of gonadotropin-dependent follicles; for both isoforms, no signal was found in most granulosa cells of ovulatory follicles. In primordial to gonadotropin-dependent follicles, cellular localization of PRLr was similar on days 0, 10 and 15 of the cycle. Oocytes consistently showed positive immunostaining for PRLrs. Comparative RT-PCR analysis of long and short PRLr expression showed that the short isoform is evenly expressed throughout the estrous cycle, whereas the expression of the long form increases at the time of estrus and decreases at mid-luteal phase and at the onset of the follicular phase. Expression of long PRLrs was greater than that of short PRLrs on day 0 of cycle; expression of both isoforms was similar on day 10 and on day 15, long PRLrs expression was lower than that of short PRLrs. Our results indicate that in sheep ovary, the maximum responsiveness to PRL might occur during the preovulatory phase of the estrous cycle. PMID:15509700

  15. Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors.

    PubMed

    Guil-Luna, S; Stenvang, J; Brünner, N; Sánchez-Céspedes, R; Millán, Y; Gómez-Laguna, J; de las Mulas, J Martín

    2014-09-01

    Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors. PMID:24249219

  16. Rational Quantitative Structure-Activity Relationship (RQSAR) Screen for PXR and CAR Isoform-Specific Nuclear Receptor Ligands

    PubMed Central

    Dring, Ann M.; Anderson, Linnea E.; Qamar, Saima; Stoner, Matthew A.

    2010-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets. PMID:20869355

  17. Immunoreactivity of progesterone receptor isoform B, nuclear factor kappaB, and IkappaBalpha in adenomyosis.

    PubMed

    Nie, Jichan; Lu, Yuan; Liu, Xishi; Guo, Sun-Wei

    2009-09-01

    Compared with normal endometrium, progesterone receptor isoform B (PR-B) and IkappaBalpha immunoreactivity were statistically significantly reduced in ectopic as well as eutopic endometrium from women with adenomyosis while nuclear p65, p50, and p52 immunoreactivity were statistically significantly increased in ectopic and eutopic endometrium. Nuclear p65 immunoreactivity was positively associated with heavier menses, and decreased PR-B and increased nuclear p65 immunoreactivity in ectopic endometrium were statistically significantly associated with the severity of dysmenorrhea in women with adenomyosis. PMID:19296948

  18. Isoforms of the CD79 signal transduction component of the macropod B-cell receptor.

    PubMed

    Suthers, Amy N; Young, Lauren J

    2014-12-01

    B cell responses and their concomitant signal transduction pathways are not well understood in marsupial mammals, despite the availability of gene expression data for key immunoglobulin genes and for elements of the CD79a/CD79b heterodimer signalling complex for two model marsupials. Broader studies of factors that influence B cell responses are still hampered by a lack of species-specific reagents and there are few reports of other factors that influence gene expression such as the potential for splice variants in BCR components, which may influence immune signalling pathways. In this study, we characterise CD79a and CD79b genes in the endangered macropod marsupial, Onychogalea fraenata (the bridled nailtail wallaby) and show that domains and residues important for the structural and functional integrity of both monomers are conserved in this species, consistent with results previously reported for the closely-related macropod, Macropus eugenii (the tammar wallaby). We extend this work to report the detection of splice variants for CD79a and CD79b in wallaby species; three CD79a isoforms and one CD79b isoform. Of these, two CD79a isoforms and the CD79b isoform have not been reported in any other mammalian species. PMID:25064685

  19. MALONDIALDEHYDE AND 4-HYDROXYNONENAL ADDUCTS ARE NOT FORMED ON CARDIAC RYANODINE RECEPTOR (RyR2) AND SARCO(ENDO)PLASMIC RETICULUM Ca2+ ATPase (SERCA2) IN DIABETES

    PubMed Central

    Moore, Caronda J.; Shao, Chun Hong; Nagai, Ryoji; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

    2013-01-01

    Recently, we reported an elevated level of glucose-derived carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca2+ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes. This study used Western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, Nε-carboxy(methyl)lysine (CML), pentosidine and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca2+ kinetics in myocytes from control, diabetic and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from control or 8 weeks diabetic rats with altered evoked Ca2+ transients. However, CML, pentosidine, and pyrraline adducts were elevated 3–5 fold (p<0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-reactive carbonyl species scavenger) reduced CML, pentosidine and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca2+ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca2+ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca2+ dysregulation. PMID:23354458

  20. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

    USGS Publications Warehouse

    Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

    2014-01-01

    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  1. Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes.

    PubMed

    Martínez-Redondo, Vicente; Jannig, Paulo R; Correia, Jorge C; Ferreira, Duarte M S; Cervenka, Igor; Lindvall, Jessica M; Sinha, Indranil; Izadi, Manizheh; Pettersson-Klein, Amanda T; Agudelo, Leandro Z; Gimenez-Cassina, Alfredo; Brum, Patricia C; Dahlman-Wright, Karin; Ruas, Jorge L

    2016-07-15

    Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators. PMID:27231350

  2. Mechanisms of Transient Signaling via Short and Long Prolactin Receptor Isoforms in Female and Male Sensory Neurons*

    PubMed Central

    Belugin, Sergei; Diogenes, Anibal R.; Patil, Mayur J.; Ginsburg, Erika; Henry, Michael A.; Akopian, Armen N.

    2013-01-01

    Prolactin (PRL) regulates activity of nociceptors and causes hyperalgesia in pain conditions. PRL enhances nociceptive responses by rapidly modulating channels in nociceptors. The molecular mechanisms underlying PRL-induced transient signaling in neurons are not well understood. Here we use a variety of cell biology and pharmacological approaches to show that PRL transiently enhanced capsaicin-evoked responses involve protein kinase C ϵ (PKCϵ) or phosphatidylinositol 3-kinase (PI3K) pathways in female rat trigeminal (TG) neurons. We next reconstituted PRL-induced signaling in a heterologous expression system and TG neurons from PRL receptor (PRLR)-null mutant mice by expressing rat PRLR-long isoform (PRLR-L), PRLR-short isoform (PRLR-S), or a mix of both. Results show that PRLR-S, but not PRLR-L, is capable of mediating PRL-induced transient enhancement of capsaicin responses in both male and female TG neurons. However, co-expression of PRLR-L with PRLR-S (1:1 ratio) leads to the inhibition of the transient PRL actions. Co-expression of PRLR-L deletion mutants with PRLR-S indicated that the cytoplasmic site adjacent to the trans-membrane domain of PRLR-L was responsible for inhibitory effects of PRLR-L. Furthermore, in situ hybridization and immunohistochemistry data indicate that in normal conditions, PRLR-L is expressed mainly in glia with little expression in rat sensory neurons (3–5%) and human nerves. The predominant PRLR form in TG neurons/nerves from rats and humans is PRLR-S. Altogether, PRL-induced transient signaling in sensory neurons is governed by PI3K or PKCϵ, mediated via the PRLR-S isoform, and transient effects mediated by PRLR-S are inhibited by presence of PRLR-L in these cells. PMID:24142695

  3. Role for the thromboxane A2 receptor β-isoform in the pathogenesis of intrauterine growth restriction

    PubMed Central

    Powell, Katie L.; Stevens, Veronica; Upton, Dannielle H.; McCracken, Sharon A.; Simpson, Ann M.; Cheng, Yan; Tasevski, Vitomir; Morris, Jonathan M.; Ashton, Anthony W.

    2016-01-01

    Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPβ) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPβ attenuated these functions and inhibited migration. Expression of the TPβ transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPβ impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans. PMID:27363493

  4. Carboxyl-Terminal Receptor Domains Control the Differential Dephosphorylation of Somatostatin Receptors by Protein Phosphatase 1 Isoforms

    PubMed Central

    Lehmann, Andreas; Kliewer, Andrea; Märtens, Jan Carlo; Nagel, Falko; Schulz, Stefan

    2014-01-01

    We have recently identified protein phosphatase 1β (PP1β) as G protein-coupled receptor (GPCR) phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ) as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs. PMID:24637622

  5. SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.

    PubMed Central

    Chen, J D; Umesono, K; Evans, R M

    1996-01-01

    Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8755515

  6. Alternative mRNA splicing of SMRT creates functional diversity by generating corepressor isoforms with different affinities for different nuclear receptors.

    PubMed

    Goodson, Michael L; Jonas, Brian A; Privalsky, Martin L

    2005-03-01

    Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts. PMID:15632172

  7. Insulin receptor isoform A and insulin-like growth factor II as additional treatment targets in human osteosarcoma.

    PubMed

    Avnet, Sofia; Sciacca, Laura; Salerno, Manuela; Gancitano, Giovanni; Cassarino, Maria Francesca; Longhi, Alessandra; Zakikhani, Mahvash; Carboni, Joan M; Gottardis, Marco; Giunti, Armando; Pollak, Michael; Vigneri, Riccardo; Baldini, Nicola

    2009-03-15

    Despite the frequent presence of an insulin-like growth factor I receptor (IGFIR)-mediated autocrine loop in osteosarcoma (OS), interfering with this target was only moderately effective in preclinical studies. Here, we considered other members of the IGF system that might be involved in the molecular pathology of OS. We found that, among 45 patients with OS, IGF-I and IGFBP-3 serum levels were significantly lower, and IGF-II serum levels significantly higher, than healthy controls. Increased IGF-II values were associated with a decreased disease-free survival. After tumor removal, both IGF-I and IGF-II levels returned to normal values. In 23 of 45 patients, we obtained tissue specimens and found that all expressed high mRNA level of IGF-II and >IGF-I. Also, isoform A of the insulin receptor (IR-A) was expressed at high level in addition to IGFIR and IR-A/IGFIR hybrids receptors (HR(A)). These receptors were also expressed in OS cell lines, and simultaneous impairment of IGFIR, IR, and Hybrid-Rs by monoclonal antibodies, siRNA, or the tyrosine kinase inhibitor BMS-536924, which blocks both IGFIR and IR, was more effective than selective anti-IGFIR strategies. Also, anti-IGF-II-siRNA treatment in low-serum conditions significantly inhibited MG-63 OS cells that have an autocrine circuit for IGF-II. In summary, IGF-II rather than IGF-I is the predominant growth factor produced by OS cells, and three different receptors (IR-A, HR(A), and IGFIR) act complementarily for an IGF-II-mediated constitutive autocrine loop, in addition to the previously shown IGFIR/IGF-I circuit. Cotargeting IGFIR and IR-A is more effective than targeting IGF-IR alone in inhibiting OS growth. PMID:19258511

  8. Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

    PubMed

    Flannery, Clare A; Rowzee, Anne M; Choe, Gina H; Saleh, Farrah L; Radford, Caitlin C; Taylor, Hugh S; Wood, Teresa L

    2016-04-01

    The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers. PMID:26862994

  9. Sex-Steroid Regulation of Relaxin Receptor Isoforms (RXFP1 & RXFP2) Expression in the Patellar Tendon and Lateral Collateral Ligament of Female WKY Rats

    PubMed Central

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and tendons of the knee. Methods: Ovariectomized adult female WKY rats were treated with different doses of estrogen (0.2, 2, 20 μg/kg), progesterone (4mg) and testosterone (125 & 250μg/kg) for three consecutive days. At the end of the treatment, the animals were sacrificed and the patellar tendon and lateral collateral ligament were harvested for mRNA and protein expression analyses by Real Time PCR and Western blotting respectively. Results: RXFP1, the main isoform expressed in these knee structures and RXFP2 showed a dose-dependent increase in expression with estrogen. Progesterone treatment resulted in an increase while testosterone caused a dose-dependent decrease in the mRNA and protein expression of both relaxin receptor isoforms. Discussion: Progesterone and high dose estrogen up-regulate while testosterone down-regulates RXFP1 and RXFP2 expression in the patellar tendon and lateral collateral ligament of rat's knee. Conclusion: Relaxin receptor isoforms up-regulation by progesterone and high dose estrogen could provide the basis for the reported increase in knee laxity while down-regulation of these receptor isoforms by testosterone could explain low incidence of non-contact knee injury in male. PMID:24465164

  10. Ryanodine-mediated conversion of STP to LTP is lacking in synaptopodin-deficient mice.

    PubMed

    Grigoryan, Gayane; Segal, Menahem

    2016-05-01

    In previous studies we and others have found that activation of ryanodine receptors (RyRs) facilitate expression of long-term potentiation (LTP) of reactivity to afferent stimulation in hippocampal slices, with a more pronounced action in the ventral hippocampus. We have also been able to link the involvement of synaptopodin (SP), an actin-binding protein, with neuronal plasticity via its interaction with RyRs. To test this link more directly, we have now compared the ability of ryanodine to convert short-term to LTP in hippocampal slices taken from normal and SP-knockout (SPKO) mice. Indeed, SPKO hippocampus expresses lower concentrations of RyRs and in slices of these mice ryanodine is unable to facilitate conversion of short-term to LTP. These observations link functionally SP with calcium stores. PMID:25772508

  11. Isoform-specific Binding of Selenoprotein P to the β-Propeller Domain of Apolipoprotein E Receptor 2 Mediates Selenium Supply*

    PubMed Central

    Kurokawa, Suguru; Bellinger, Frederick P.; Hill, Kristina E.; Burk, Raymond F.; Berry, Marla J.

    2014-01-01

    Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery. PMID:24532792

  12. Localization of the mRNAs for two dopamine D2 receptor isoforms in the rat brain.

    PubMed

    Fujiwara, Y; Sora, I; Tomita, H; Akiyama, K; Otsuki, S; Yamamura, H I

    1991-12-01

    Two molecular forms of the dopamine D2 receptor were generated by alternative RNA splicing. To investigate the relative distributions of the two mRNAs encoding the D2 receptor isoforms, D2(415) and D2(444), we performed in situ hybridization histochemistry in the rat brain with the two oligonucleotide probes. An insert probe complementary to an additional fragment of the D2 receptor mRNA cloned from the rat brain, and a spanning probe complementary to its contiguous sequence were used. These 48 base probes were 3'-end labeled with [35S]dATP. The brains were dissected from male SD rats and frozen in dry ice and acetone. Cryostat sections (16 microns) were collected on gelatin coated slides and stored at -20 degrees C. In situ hybridization studies were conducted with a probe concentration of 1 x 10(6) dpm/100 microliters of buffer per brain slice at 37 degrees C for 18-20 h in a humid chamber. The slides were washed, dried and exposed to tritium sensitive film for one week. The autoradiograph showed that both mRNA were present at high levels in the corpus striatum, accumbens nucleus and substantia nigra (pars compacta). Identical patterns of labeling were obtained in the rat brain using both the insert and spanning probes, although the optical densities detected with the insert probe were higher than those with the spanning probe in the corpus striatum. This suggests that both D2 receptor mRNAs are expressed similarly in each region of the rat brain and D2(444) expressed dominantly in the corpus striatum. PMID:1813683

  13. The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells.

    PubMed

    Herd, Murray B; Haythornthwaite, Alison R; Rosahl, Thomas W; Wafford, Keith A; Homanics, Gregg E; Lambert, Jeremy J; Belelli, Delia

    2008-02-15

    The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this

  14. Isoform-selective physical coupling of TRPC3 channels to IP3 receptors in smooth muscle cells regulates arterial contractility

    PubMed Central

    Adebiyi, Adebowale; Zhao, Guiling; Narayanan, Damodaran; Thomas, Candice M.; Bannister, John P.; Jaggar, Jonathan H.

    2010-01-01

    Rationale Inositol 1,4,5-trisphosphate (IP3)-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives Study mechanisms by which IP3Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Methods and Results Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C-coupled receptor agonist, elevated immuno-FRET between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, co-immunoprecipitated with IP3R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP3-induced non-selective cation current (ICat) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP3R1 and TRPC3, IP3-induced ICat activation, and ET-1 and IP3-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP3R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R N-terminal region that can interact with TRPC channels activated ICat. A TRPC3 CIRB domain peptide attenuated IP3- and ET-1-induced ICat activation and vasoconstriction. Conclusions IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction. PMID:20378853

  15. The Ontogeny of Nuclear Estrogen Receptor Isoform Expression and the Effect of 17β Estradiol in Embryonic Rainbow Trout (Oncorhynchus mykiss)

    PubMed Central

    Boyce-Derricott, Josh; Nagler, James J.; Cloud, J.G.

    2009-01-01

    Ligand bound nuclear estrogen receptor (ER) acts as a transcription factor regulating the expression of estrogen dependent genes. There are four nuclear ER isoforms in rainbow trout (Oncorhynchus mykiss). The objective of this study was to measure whole body mRNA levels of the two ERα isoforms (α1/α2) and the two ERβ isoforms (β1/β2) in male and female embryos from 50 to 600 degree-days (DD; days post-fertilization x water temperature) and in embryos exposed to vehicle or 17β-estradiol E2) for 2 hours at 230, 240 and 250 DD. All four isoforms were detected at every time point in both sexes. Sexual dimorphism was rarely observed; at 50 DD the level of ERα2 mRNA was significantly greater in males than in females and at 100 DD the level of ERβ1 mRNA was significantly greater in females than in males (p<0.05). Expression profiles of the two ERα isoforms were slightly different from one another, whereas the ERβ isoforms exhibited similar expression patterns. The effect of E2 was not different between male and female embryos. The level of ERα1 mRNA increased significantly at 240 DD; a similar but not statistically significant trend was observed at 230 and 250 DD. Despite the critical role of estrogen during sex differentiation in rainbow trout, the receptivity to this hormone as measured by the response in mRNA levels of ER appears to be largely the same between males and females and ERα1 is the only E2 responsive isoform. PMID:19818378

  16. Neuromedin U directly stimulates growth of cultured rat calvarial osteoblast-like cells acting via the NMU receptor 2 isoform.

    PubMed

    Rucinski, Marcin; Ziolkowska, Agnieszka; Tyczewska, Marianna; Szyszka, Marta; Malendowicz, Ludwik K

    2008-09-01

    The neuromedin U (NMU) system is composed of NMU, neuromedin S (NMS) and their receptors NMUR1 and NMUR2. This system is involved in the regulation of energy homeostasis, neuroendocrine functions, immune response, circadian rhythm and spermatogenesis. The present study aimed to investigate the possible role of the NMU system in regulating functions of cultured rat calvarial osteoblast-like (ROB) cells. By using QPCR, high expression of NMU mRNA was found in freshly isolated ROB cells while after 7, 14, and 21 days of culture, expression of the studied gene was very low. In contrast, NMUR2 mRNA expression in freshly isolated ROB cells was negligible and very high in cultured cells. The highest NMUR2 mRNA expression was observed at day 7, and was followed by lower levels at days 14 and 21 of culture. Neither NMS nor NMUR1 mRNA was found in studied cells. Exposure of cultured ROB cells to NMU8 at concentrations 10(-6) to 10(-10) M had no effect on expression levels of the genes. During the entire culture period, NMU8 did not affect osteocalcin production, but stimulated proliferative activity of ROB cells at days 14 and 21 of culture. Thus, we demonstrated that cultured rat calvarial osteoblast-like cells are provided with NMUR2, the receptor isoform typical for the central nervous system. Acting via this receptor NMU8 stimulates proliferation of cultured cells and has no effect on their differentiated function (osteocalcin secretion). PMID:18698496

  17. Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production.

    PubMed

    Morgan, David J; Poolman, Toryn M; Williamson, Andrew J K; Wang, Zichen; Clark, Neil R; Ma'ayan, Avi; Whetton, Anthony D; Brass, Andrew; Matthews, Laura C; Ray, David W

    2016-01-01

    The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function. PMID:27226058

  18. Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production

    PubMed Central

    Morgan, David J.; Poolman, Toryn M.; Williamson, Andrew J. K.; Wang, Zichen; Clark, Neil R.; Ma’ayan, Avi; Whetton, Anthony D.; Brass, Andrew; Matthews, Laura C.; Ray, David W.

    2016-01-01

    The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function. PMID:27226058

  19. Differential α4(+)/(-)β2 Agonist-binding Site Contributions to α4β2 Nicotinic Acetylcholine Receptor Function within and between Isoforms.

    PubMed

    Lucero, Linda M; Weltzin, Maegan M; Eaton, J Brek; Cooper, John F; Lindstrom, Jon M; Lukas, Ronald J; Whiteaker, Paul

    2016-01-29

    Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define function/nAChR. If the α4(+)/(-)β2 sites contribute equally to function, making identical β2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect. PMID:26644472

  20. Negative Effects of SRD5A1 on Nuclear Activity of Progesterone Receptor Isoform B in JEG3 Cells.

    PubMed

    Miao, Zhuo; Sun, Min; Jiang, Feng; Yao, Yuanqing; Li, Yi

    2016-02-01

    Progesterone withdrawal signals labor in mammals. Elevated intracellular metabolism contributes to progesterone functional withdrawal through unknown mechanism, which is thought to act via progesterone receptor (PR). This study aims to investigate molecular mechanisms underlying progesterone withdrawal during pregnancy and labor. We investigated the role of 5α-reductase type I (SRD5A1) in enzymatic catalysis of progesterone and loss of PR function in a human trophoblast choriocarcinoma cell line JEG3. The PR isoform B (PR-B) was robustly expressed in JEG3 cells. The SRD5A1 small-interfering RNA knockdown led to significant increase in PR-B nuclear import, ectopic, whereas SRD5A1 overexpression resulted in remarkable inhibition of nuclear PR-B in P4-treated cells. Repression of SRD5A1 activated PR-B responsive gene, whereas overexpression of SRD5A1 possessed an inhibitory effect. JEG3 cell line is a valuable tool to study mechanisms responsible for loss of PR function and screening of drugs for preterm birth treatment. Our study aims to investigate the molecular mechanisms underlying progesterone withdrawal during pregnancy and labor. PMID:26243543

  1. Progestins reinitiate cell cycle progression in antiestrogen-arrested breast cancer cells through the B-isoform of progesterone receptor.

    PubMed

    McGowan, Eileen M; Russell, Amanda J; Boonyaratanakornkit, Viroj; Saunders, Darren N; Lehrbach, Gillian M; Sergio, C Marcelo; Musgrove, Elizabeth A; Edwards, Dean P; Sutherland, Robert L

    2007-09-15

    Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest. PMID:17875737

  2. The Ability of Thyroid Hormone Receptors to Sense T4 as an Agonist Depends on Receptor Isoform and on Cellular Cofactors

    PubMed Central

    Schroeder, Amy; Jimenez, Robyn; Young, Briana

    2014-01-01

    T4 (3,5,3′,5′-tetraiodo-l-thyronine) is classically viewed as a prohormone that must be converted to the T3 (3,5,3′-triiodo-l-thyronine) form for biological activity. We first determined that the ability of reporter genes to respond to T4 and to T3 differed for the different thyroid hormone receptor (TR) isoforms, with TRα1 generally more responsive to T4 than was TRβ1. The response to T4 vs T3 also differed dramatically in different cell types in a manner that could not be attributed to differences in deiodinase activity or in hormone affinity, leading us to examine the role of TR coregulators in this phenomenon. Unexpectedly, several coactivators, such as steroid receptor coactivator-1 (SRC1) and thyroid hormone receptor-associated protein 220 (TRAP220), were recruited to TRα1 nearly equally by T4 as by T3 in vitro, indicating that TRα1 possesses an innate potential to respond efficiently to T4 as an agonist. In contrast, release of corepressors, such as the nuclear receptor coreceptor NCoRω, from TRα1 by T4 was relatively inefficient, requiring considerably higher concentrations of this ligand than did coactivator recruitment. Our results suggest that cells, by altering the repertoire and abundance of corepressors and coactivators expressed, may regulate their ability to respond to T4, raising the possibility that T4 may function directly as a hormone in specific cellular or physiological contexts. PMID:24673558

  3. Retinoic acid receptor-related orphan receptor (ROR) alpha4 is the predominant isoform of the nuclear receptor RORalpha in the liver and is up-regulated by hypoxia in HepG2 human hepatoma cells.

    PubMed Central

    Chauvet, Caroline; Bois-Joyeux, Brigitte; Danan, Jean-Louis

    2002-01-01

    The retinoic acid receptor-related orphan receptor alpha (RORalpha) is critically involved in many physiological functions in several organs. We find that the main RORalpha isoform in the mouse liver is the RORalpha4 isoform, in terms of both mRNA and protein levels, while the RORalpha1 isoform is less abundant. Because hypoxia is a major feature of liver physiology and pathology, we examined the effect of this stress on Rora gene expression and RORalpha transcriptional activity. HepG2 human hepatoma cells were cultured for 24 h under normoxia (20% O2) or hypoxia (10, 2, and 0.1% O2) and the abundance of the Rora transcripts measured by Northern blot and semi-quantitative RT-PCR. Hypoxic HepG2 cells contained more Rora mRNA than controls. This was also observed in rat hepatocytes in primary culture. Cobalt chloride and desferrioxamine also increased the amount of Rora mRNA in HepG2 cells. It is likely that these treatments increase the amount of the RORalpha4 protein in HepG2 cells as evidenced by Western blotting in the case of desferrioxamine. Transient transfection experiments indicated that hypoxia, cobalt chloride, and desferrioxamine all stimulate RORalpha transcriptional activity in HepG2 cells. Hence, we believe that RORalpha participates in the control of gene transcription in hepatic cells and modulates gene expression in response to hypoxic stress. PMID:12023888

  4. Differential Interactions of Specific Nuclear Factor I Isoforms with the Glucocorticoid Receptor and STAT5 in the Cooperative Regulation of WAP Gene Transcription

    PubMed Central

    Mukhopadhyay, Sudit S.; Wyszomierski, Shannon L.; Gronostajski, Richard M.; Rosen, Jeffrey M.

    2001-01-01

    The distal region (−830 to −720 bp) of the rat whey acidic protein (WAP) gene contains a composite response element (CoRE), which has been demonstrated previously to confer mammary gland-specific and hormonally regulated WAP gene expression. Point mutations in the binding sites for specific transcription factors present within this CoRE have demonstrated the importance of both nuclear factor I (NFI) and STAT5 as well as cooperative interactions with the glucocorticoid receptor (GR) in the regulation of WAP gene expression in the mammary gland of transgenic mice. This study reports the characterization of NFI gene expression during mammary gland development and the identification and cloning of specific NFI isoforms (NFI-A4, NFI-B2, and NFI-X1) from the mouse mammary gland during lactation. Some but not all of these NFI isoforms synergistically activate WAP gene transcription in cooperation with GR and STAT5, as determined using transient cotransfection assays in JEG-3 cells. On both the WAP CoRE and the mouse mammary tumor virus long terminal repeat promoter, the NFI-B isoform preferentially activated gene transcription in cooperation with STAT5A and GR. In contrast, the NFI-A isoform suppressed GR and STAT cooperativity at the WAP CoRE. Finally, unlike their interaction with the NFI consensus binding site in the adenovirus promoter, the DNA-binding specificities of the three NFI isoforms to the palindromic NFI site in the WAP CoRE were not identical, which may partially explain the failure of the NFI-A isoform to cooperate with GR and STAT5A. PMID:11564870

  5. Differential expression of estrogen receptor α and β isoforms in multiple and solitary leiomyomas.

    PubMed

    Shao, Ruyue; Fang, Liaoqiong; Xing, Ruoxi; Xiong, Yu; Fang, Liaoqiong; Wang, Zhibiao

    Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E2) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E2 concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes. PMID:26529545

  6. MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

  7. ApoE-isoform-dependent cellular uptake of amyloid-β is mediated by lipoprotein receptor LR11/SorLA.

    PubMed

    Yajima, Ryuji; Tokutake, Takayoshi; Koyama, Akihide; Kasuga, Kensaku; Tezuka, Toshiyuki; Nishizawa, Masatoyo; Ikeuchi, Takeshi

    2015-01-01

    The formation of senile plaques composed of β-amyloid (Aβ) in the brain is likely the initial event in Alzheimer's disease (AD). Possession of the APOE ε4 allele, the strong genetic factor for AD, facilitates the Aβ deposition from the presymptomatic stage of AD in a gene-dosage-dependent manner. However, the precise mechanism by which apoE isoforms differentially induce the AD pathology is largely unknown. LR11/SorLA is a type I membrane protein that functions as the neuronal lipoprotein endocytic receptor of apoE and the sorting receptor of the amyloid precursor protein (APP) to regulate amyloidogenesis. Recently, LR11/SorLA has been reported to be involved in the lysosomal targeting of extracellular amyloid-β (Aβ) through the binding of Aβ to the vacuolar protein sorting 10 (VPS10) protein domain of LR11/SorLA. Here, we attempted to examine the human-apoE-isoform-dependent effect on the cellular uptake of Aβ through the formation of a complex between an apoE isoform and LR11/SorLA. Cell culture experiments using Neuro2a cells revealed that the cellular uptake of secreted apoE3 and apoE4 was enhanced by the overexpression of LR11/SorLA. In contrast, the cellular uptake of apoE2 was not affected by the expression of LR11/SorLA. Co-immunoprecipitation assay revealed that apoE-isoform-dependent differences were observed in the formation of an apoE-LR11 complex (apoE4>apoE3>apoE2). ApoE-isoform-dependent differences in cellular uptake of FAM-labeled Aβ were further investigated by coculture assay in which donor cells secrete one of the apoE isoforms and recipient cells express FL-LR11. The cellular uptake of extracellular Aβ into the recipient cells was most prominently accentuated when cocultured with the donor cells secreting apoE4 in the medium, followed by apoE3 and apoE2. Taken together, our results provide evidence for the mechanism whereby human-apoE-isoform-dependent differences modulate the cellular uptake of Aβ mediated by LR11/SorLA. PMID

  8. Anti-metastatic outcome of isoform-specific prolactin receptor targeting in breast cancer.

    PubMed

    Yonezawa, Tomohiro; Chen, Kuan-Hui Ethan; Ghosh, Mrinal K; Rivera, Lorena; Dill, Riva; Ma, Lisa; Villa, Pedro A; Kawaminami, Mitsumori; Walker, Ameae M

    2015-09-28

    Controversy exists concerning the role of the long prolactin receptor (PRLR) in the progression of breast cancer. By targeting pre-mRNA splicing, we succeeded in knocking down only the long PRLR in vivo, leaving the short forms unaffected. Using two orthotopic and highly-metastatic models of breast cancer, one of which was syngeneic (mouse 4T1) to allow assessment of tumor-immune interactions and one of which was endocrinologically humanized (human BT-474) to activate human PRLRs, we examined the effect of long PRLR knockdown on disease progression. In both models, knockdown dramatically inhibited metastatic spread to the lungs and liver and resulted in increased central death in the primary tumor. In the syngeneic model, immune infiltrates in metastatic sites were changed from innate inflammatory cells to lymphocytes, with an increase in the incidence of tumor-specific cytotoxic T cells. Long PRLR knockdown in three-dimensional culture induced apoptosis of tumor-initiating/cancer stem cells (death of 95% of cells displaying stem cell markers in 15 days). We conclude that the long PRLR plays an important role in breast cancer metastasis. PMID:26095602

  9. The Short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction

    PubMed Central

    Chen, Lanfen; Chen, Zhangguo; Baker, Kristi; Halvorsen, E lizabeth M.; da Cunha, Andre Pires; Flak, Magdalena B.; Gerber, Georg; Huang, Yu-Hwa; Hosomi, Shuhei; Arthur, J anelle C.; Dery, Ken J.; Nagaishi, Takashi; Beauchemin, Nicole; Holmes, Kathryn V.; Ho, Joshua W. K.; Shively, John E.; Jobin, Christian; Onderdonk, Andrew B.; Bry, Lynn; Weiner, Howard L.; Higgins, Darren E.; Blumberg, Richard S.

    2012-01-01

    Summary Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens. PMID:23123061

  10. Regulation of carnitine palmitoyltransferase I (CPT-Iα) gene expression by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) isoforms

    PubMed Central

    Sadana, Prabodh; Zhang, Yi; Song, Shulan; Cook, George A.; Elam, Marshall B.; Park, Edwards A.

    2007-01-01

    Summary The peroxisome proliferator-activated receptor gamma coactivators (PGC-1) have important roles in mitochondrial biogenesis and metabolic control in a variety of tissues. There are multiple isoforms of PGC-1 including PGC-1α and PGC-1β. Both the PGC-1α and β isoforms promote mitochondrial biogenesis and fatty acid oxidation, but only PGC-1α stimulates gluconeogenesis in the liver. Carnitine palmitoyltransferase I (CPT-I) is a key enzyme regulating mitochondrial fatty acid oxidation. In these studies, we determined that PGC-1β stimulated expression of the “liver” isoform of CPT-I (CPT-Iα) but that PGC-1β did not induce pyruvate dehydrogenase kinase 4 (PDK4) which is a regulator of pyruvate metabolism. The CPT-Iα gene is induced by thyroid hormone. We found that T3 increased the expression of PGC-1β and that PGC-1β enhanced the T3 induction of CPT-Iα. The thyroid hormone receptor interacts with PGC-1β in a ligand dependent manner. Unlike PGC-1α, the interaction of PGC-1β and the T3 receptor does not occur exclusively through the leucine-X-X-leucine-leucine motif in PGC-1β. We have found that PGC-1β is associated with the CPT-Iα gene in vivo. Overall, our results demonstrate that PGC-1β is a coactivator in the T3 induction of CPT-Iα and that PGC-1β has similarities and differences with the PGC-1α isoform. PMID:17239528

  11. Differential expression of neuregulin-1 isoforms and downregulation of erbin are associated with Erb B2 receptor activation in diabetic peripheral neuropathy

    PubMed Central

    2013-01-01

    Background Aberrant neuron/glia interactions can contribute to a variety of neurodegenerative diseases and we have previously demonstrated that enhanced activation of Erb B2, which is a member of the epidermal growth factor receptor (EGFR) family, can contribute to the development of diabetic peripheral neuropathy (DPN). In peripheral nerves, Erb B receptors are activated by various members of the neuregulin-1 (NRG1) family including NRG1 Type I, NRG1 Type II and NRG1 Type III to regulate Schwann cell (SC) growth, migration, differentiation and dedifferentiation. Alternatively, Erb B2 activity can be negatively regulated by association with the Erb B2-interacting protein, erbin. Since the effect of diabetes on the expression of NRG1 isoforms and erbin in peripheral nerve are unknown, the current study determined whether changes in NRG1 isoforms and erbin may be associated with altered Erb B2 signaling in DPN. Results Swiss Webster mice were rendered diabetic with streptozotocin (STZ) and after 12 weeks of diabetes, treated with erlotinib, an inhibitor of Erb B2 activation. Inhibition of Erb B2 signaling partially reversed several pathophysiologic aspects of DPN including a pronounced sensory hypoalgesia, nerve conduction velocity deficits and the decrease in epidermal nerve fiber innervation. We also observed a decrease of NRG1 Type III but an increase of NRG1 Type I level in diabetic sural nerves at early stage of diabetes. With disease progression, we detected reduced erbin expression and enhanced MAPK pathway activity in diabetic mice. Inhibition of Erb B2 receptor suppressed MAPK pathway activity in treated-diabetic sural nerves. Conclusions These results support that hyperglycemia may impair NRG1/Erb B2 signaling by disrupting the balance between NRG1 isoforms, decreasing the expression of erbin and correspondingly activating the MAPK pathway. Together, imbalanced NRG1 isoforms and downregulated erbin may contribute to the dysregulation of Erb B2 signaling in

  12. The T3 receptor beta1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes.

    PubMed

    Martinez de Mena, Raquel; Scanlan, Thomas S; Obregon, Maria-Jesus

    2010-10-01

    Brown adipose tissue (BAT) thermogenesis increases when uncoupling protein-1 (UCP1) is activated adrenergically and requires T3. In humans, UCP1 activation in BAT seems involved in body weight maintenance. BAT type 2 deiodinase (D2) increases in response to adrenergic agents, producing the T3 required for UCP1 expression. T3 actions are mediated by thyroid hormone nuclear T3 receptors (TR), TRα and TRβ. Studies in mice suggest that TRβ is required for UCP1 induction, whereas TRα regulates body temperature and adrenergic sensitivity. In the present study, we compare the effects of T3 vs. specific TRβ1 and TRα1 agonists [GC-1 and CO23] on the adrenergic induction of UCP1 and D2 in cultured rat brown adipocytes. T3 and GC-1 produced similar increases on UCP1, whereas CO23 increased UCP1 only at high doses (50 nm). GC-1 at low doses (0.2-10 nm) was less potent than T3, increasing the adrenergic stimulation of D2 activity and mRNA. At higher doses, GC-1 further stimulated whereas T3 inhibited D2 activity but not D2 mRNA, suggesting posttranscriptional effects. CO23 had no effect on D2 activity but increased D2 mRNA. T3, GC-1, or CO23 by themselves did not increase UCP1 or D2 mRNA. High T3 doses shortened D2 half-life and increased D2 turnover via proteasome, whereas GC-1 did not change D2 stability. The α1- and α2-adrenergic D2 responses increased using high T3 doses. In summary, T3 increases the adrenergic stimulation of UCP1 and D2 expression mostly via the TRβ1 isoform, and in brown adipocytes, D2 is protected from degradation by the action of T3 on TRβ1. PMID:20719854

  13. Ternary Complex of Transforming Growth Factor-[beta]1 Reveals Isoform-specific Ligand Recognition and Receptor Recruitment in the Superfamily

    SciTech Connect

    Radaev, Sergei; Zou, Zhongcheng; Huang, Tao; Lafer, Eileen M.; Hinck, Andrew P.; Sun, Peter D.

    2010-11-03

    Transforming growth factor (TGF)-{beta}1, -{beta}2, and -{beta}3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-{angstrom} resolution crystal structure of the ternary complex between human TGF-{beta}1 and the extracellular domains of its type I and type II receptors, T{beta}RI and T{beta}RII. The TGF-{beta}1 ternary complex structure is similar to previously reported TGF-{beta}3 complex except with a 10{sup o} rotation in T{beta}RI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-{beta}. T{beta}RI showed significant binding to TGF-{beta}2 and TGF-{beta}3 but not TGF-{beta}1, and the binding to all three isoforms of TGF-{beta} was enhanced considerably in the presence of T{beta}RII. The preference of TGF-{beta}2 to T{beta}RI suggests a variation in its receptor recruitment in vivo. Although TGF-{beta}1 and TGF-{beta}3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-{beta} superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of T{beta}RII on TGF-{beta} is unique to TGF-{beta}s, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.

  14. Placenta expressing the greatest quantity of bisphenol A receptor ERR{gamma} among the human reproductive tissues: Predominant expression of type-1 ERRgamma isoform.

    PubMed

    Takeda, Yukimasa; Liu, Xiaohui; Sumiyoshi, Miho; Matsushima, Ayami; Shimohigashi, Miki; Shimohigashi, Yasuyuki

    2009-07-01

    Estrogen-related receptor gamma (ERRgamma), one of the 48 human nuclear receptors, has a fully active conformation with no ligand. We recently demonstrated that ERRgamma binds strongly bisphenol A (BPA), one of the nastiest endocrine disruptors, and thus retaining ERRgamma's high basal constitutive activity. A report that BPA accumulates in the human maternal-fetal placental unit has led us to hypothesize that a large amount of ERRgamma might exist in the human placenta. Here we report evidence that placenta indeed expresses ERRgamma exceptionally strongly. We first ascertained the presence of nine different ERRgamma mRNA variants and the resulting three ERRgamma protein isoforms. By real-time PCR, we estimated the relative amount of ERRgamma mRNA using total RNA extracts from human reproductive tissues. Placenta was found to express ERRgamma extremely highly. Among the three ERRgamma protein isoforms, placenta exclusively expresses the type-1 isoform, which possesses additional 23-mer amino-acid residues at the N-terminus of the ordinary ERRgamma. This N-terminal elongation was found to elevate by approximately 50% the basal constitutive activity of ERRgamma, as evidenced in the luciferase reporter gene assay. The present results suggest that BPA accumulates in the placenta by binding to ERRgamma. PMID:19304792

  15. Molecular and functional characterization of a novel aryl hydrocarbon receptor isoform, AHR1β, in the chicken (Gallus gallus).

    PubMed

    Lee, Jin-Seon; Iwabuchi, Kohei; Nomaru, Koji; Nagahama, Nobumasa; Kim, Eun-Young; Iwata, Hisato

    2013-12-01

    Dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause toxic effects through activation of the aryl hydrocarbon receptor (AHR)-mediated signaling pathway. Our previous studies have investigated the function of 2 AHR isoforms (AHR1 and AHR2) in avian species and identified a third AHR in the chicken (Gallus gallus) genome. Knowledge of multiple avian AHRs is indispensable to understand molecular mechanisms of AHR-mediated toxic effects and establish risk assessment framework for environmental AHR ligands in avian species. In this study, we successfully isolated a third novel AHR1-like cDNA from chicken and designated it as chicken AHR1 beta (ckAHR1β). The mRNA expression of ckAHR1β was primarily detected in the liver, and the hepatic protein expression was confirmed by Western blotting. Although mRNA expression of ckAHR1β was not altered by in ovo TCDD exposure, ckAHR1β exhibited specific binding to [(3)H]TCDD, TCDD-dependent nuclear translocation, and interaction with xenobiotic responsive elements (XREs) and AHR nuclear translocators (ARNTs). In vitro XRE-driven reporter gene assays revealed ckAHR1β-mediated transactivation of TCDD in a dose-dependent manner, showing a 10-fold reduced sensitivity (high EC50) compared with that mediated by ckAHR1. The mutation of Val(371) to Ser(371) in the ligand-binding domain of ckAHR1β shifted the TCDD-EC50 toward the value observed in ckAHR1, indicating the critical roles of the amino acid in sensitivity. Furthermore, ckAHR1β-mediated transactivation of TCDD was enhanced by 17β-estradiol (E2)-activated chicken estrogen receptor α (ckERα), suggesting a positive cross talk between ckERα and ckAHR1β signaling pathway. Both TCDD-induced and its enhanced activities by E2 were suppressed by the ckAHR repressor in a manner similar to ckAHR1. Collectively, our findings discover the role of ckAHR1β in dioxin toxicity and give an insight into the evolutionary history of the AHR signaling pathway. PMID:23997109

  16. Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes.

    PubMed

    Rondini, Elizabeth A; Duniec-Dmuchowski, Zofia; Kocarek, Thomas A

    2016-04-01

    Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism. PMID:26798158

  17. Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery, Sex and Betamethasone Exposure

    PubMed Central

    Saif, Zarqa; Dyson, Rebecca M.; Palliser, Hannah K.; Wright, Ian M. R.; Lu, Nick; Clifton, Vicki L.

    2016-01-01

    The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms. PMID:26840867

  18. Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery, Sex and Betamethasone Exposure.

    PubMed

    Saif, Zarqa; Dyson, Rebecca M; Palliser, Hannah K; Wright, Ian M R; Lu, Nick; Clifton, Vicki L

    2016-01-01

    The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms. PMID:26840867

  19. Vascular endothelial growth factor-A isoform and (co)receptor expression are differentially regulated by 17beta-oestradiol in the ovariectomised mouse uterus.

    PubMed

    Walter, Lisa M; Rogers, Peter A W; Girling, Jane E

    2010-08-01

    The angiogenic effects of 17beta-oestradiol (E(2)) in the mouse endometrium are mediated by vascular endothelial growth factor-A (VEGFA). We analysed the temporal and spatial changes in VEGFA isoform and (co)receptor expression in ovariectomised mouse uteri following E(2) treatment. VEGFA isoform and receptor mRNA were quantified in whole uterine tissue collected 2, 6, 12 and 24 h after E(2) or vehicle treatment. Laser capture microdissection was used to investigate mRNA expression in epithelial, stromal and myometrial tissues separately. Endothelial cell proliferation, VEGFA and VEGF receptor-2 (VEGFR2) protein were visualised using immunohistochemistry. Endometrial endothelial cell proliferation was only observed 24 h after E(2) treatment. In whole uterine tissue, total Vegfa, Vegfa(164) and Vegfa(120) mRNA expression increased 2 h post E(2) treatment, and then decreased by 24 h. Vegfa(188) expression was lower in E(2)-treated animals at all time points relative to control animals. Vegfr2 and neuropilin-1 (Nrp1) mRNA expression did not change following E(2) treatment; Nrp2 expression decreased by 24 h. When uterine compartments were considered separately at 24 h post E(2) or vehicle, stromal Vegfa(120), Vegfa(188) and Vegfr2 mRNA expression and myometrial Vegfa(120) and Vegfa(188) mRNA expression were reduced in E(2)-treated mice relative to controls, whereas epithelial Vegfa(188) mRNA expression increased. The highest VEGFA immunoexpression was observed in luminal epithelium; expression increased at 24 h relative to other time points. No changes were noted in VEGFR2 immunoexpression among treatment groups. We have provided the first evidence that VEGFA isoform and receptor mRNA expression are differentially regulated by E(2) in different uterine cell compartments. PMID:20530092

  20. Testosterone Reduces Knee Passive Range of Motion and Expression of Relaxin Receptor Isoforms via 5α-Dihydrotestosterone and Androgen Receptor Binding

    PubMed Central

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity. PMID:24642882

  1. Testosterone reduces knee passive range of motion and expression of relaxin receptor isoforms via 5α-dihydrotestosterone and androgen receptor binding.

    PubMed

    Dehghan, Firouzeh; Muniandy, Sekaran; Yusof, Ashril; Salleh, Naguib

    2014-01-01

    Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity. PMID:24642882

  2. Band 4.1 proteins are expressed in the retina and interact with both isoforms of the metabotropic glutamate receptor type 8.

    PubMed

    Rose, Melanie; Dütting, Eva; Enz, Ralf

    2008-06-01

    The function of the CNS depends on the correct regulation of neurotransmitter receptors by interacting proteins. Here, we screened a retinal cDNA library for proteins interacting with the intracellular C-terminus of the metabotropic glutamate receptor isoform 8a (mGluR8a). The band 4.1B protein binds to the C-termini of mGluR8a and mGluR8b, co-localizes with these glutamate receptors in transfected mammalian cells, facilitates their cell surface expression and inhibits the mGluR8 mediated reduction of intracellular cAMP concentrations. In contrast, no interaction with 4.1B was observed for other mGluRs tested. Amino acids encoded by exons 19 and 20 of 4.1B and a stretch of four basic amino acids present in the mGluR8 C-termini mediate the protein interaction. Besides binding to 4.1B, mGluR8 isoforms interact with 4.1G, 4.1N, and 4.1R. Because band 4.1 transcripts undergo extensive alternative splicing, we analyzed the splicing pattern of interacting regions and detected a 4.1B isoform expressed specifically in the retina. Within this tissue, mGluR8 and 4.1B, 4.1G, 4.1N, and 4.1R show a comparable distribution, being expressed in both synaptic layers and in somata of the ganglion cell layer. In summary, our studies identified band 4.1 proteins as new players for the mGluR8 mediated signal transduction. PMID:18373558

  3. Novel, isoform-selective, cholecystokinin A receptor antagonist inhibits colon and pancreatic cancers in preclinical models through novel mechanism of action.

    PubMed

    Ponnusamy, Suriyan; Lattmann, Eric; Lattmann, Pornthip; Thiyagarajan, Thirumagal; Padinjarethalakal, Balaram N; Narayanan, Ramesh

    2016-04-01

    Colon and pancreatic cancers contribute to 90,000 deaths each year in the USA. These cancers lack targeted therapeutics due to heterogeneity of the disease and multiple causative factors. One important factor that contributes to increased colon and pancreatic cancer risk is gastrin. Gastrin mediates its actions through two G-protein coupled receptors (GPCRs): cholecystokinin receptor A (CCK-A) and CCK-B/gastrin receptor. Previous studies have indicated that colon cancer predominantly expresses CCK-A and responds to CCK-A isoform antagonists. However, many CCK-A antagonists have failed in the clinic due to poor pharmacokinetic properties or lack of efficacy. In the present study, we synthesized a library of CCK-A isoform-selective antagonists and tested them in various colon and pancreatic cancer preclinical models. The lead CCK-A isoform, selective antagonist PNB-028, bound to CCK-A at 12 nM with a 60-fold selectivity towards CCK-A over CCK-B. Furthermore, it inhibited the proliferation of CCK-A-expressing colon and pancreatic cancer cells without affecting the proliferation of non-cancerous cells. PNB-028 was also extremely effective in inhibiting the growth of MAC-16 and LoVo colon cancer and MIA PaCa pancreatic cancer xenografts in immune-compromised mice. Genome‑wide microarray and kinase-array studies indicate that PNB-028 inhibited oncogenic kinases and angiogenic factors to inhibit the growth of colon cancer xenografts. Safety pharmacology and toxicology studies have indicated that PNB-028 is extremely safe and has a wide safety margin. These studies suggest that targeting CCK-A selectively renders promise to treat colon and pancreatic cancers and that PNB-028 could become the next-generation treatment option. PMID:26820391

  4. Changes in progesterone receptor isoforms expression and in the morphology of the oviduct magnum of mature laying and aged nonlaying hens.

    PubMed

    González-Morán, María Genoveva

    2016-09-16

    The aim of this study was to evaluate changes in the morphology and expression of progesterone receptor (PR) isoforms in different cell subpopulations of the magnum region of the left oviduct of mature laying and aged nonlaying hens through histomorphometric and immunohistological methods. Histological results demonstrated several changes in the oviduct magnum of mature and aged hens, mainly in the mucosal tissue. Immunohistochemical analysis showed that both PR isoforms are expressed in all cell types of the oviduct magnum of mature laying hens. In contrast, in each cell type of the oviduct magnum of aged nonlaying hens only one PR isoform (PR-A or PR-B) was expressed. The results indicate that PR percentage and the PR-A and PR-B ratio change according to the cell type of the oviduct magnum and in an age-specific manner, and suggest that these variations contribute to the regulation of progesterone actions in the oviduct magnum with the normal aging of the animal. PMID:27526993

  5. Reduced Peripheral Expression of the Glucocorticoid Receptor α Isoform in Individuals with Posttraumatic Stress Disorder: A Cumulative Effect of Trauma Burden

    PubMed Central

    Morath, Julia; Adenauer, Hannah; Elbert, Thomas; Engler, Harald

    2014-01-01

    Background Posttraumatic stress disorder (PTSD) is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA) axis and changes in glucocorticoid (GC) responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR) of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. Methods GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42) as well as in ethnically matched reference subjects (non-PTSD, n = 35). Results Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. Conclusions Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences. PMID:24466032

  6. Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

    PubMed Central

    1993-01-01

    The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that

  7. Differential Regulation of Breast Cancer-Associated Genes by Progesterone Receptor Isoforms PRA and PRB in a New Bi-Inducible Breast Cancer Cell Line

    PubMed Central

    Khan, Junaid A.; Bellance, Catherine; Guiochon-Mantel, Anne; Lombès, Marc; Loosfelt, Hugues

    2012-01-01

    Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB. PMID:23029355

  8. Amino Acid Residues 489–503 of Dihydropyridine Receptor (DHPR) β1a Subunit Are Critical for Structural Communication between the Skeletal Muscle DHPR Complex and Type 1 Ryanodine Receptor*

    PubMed Central

    Eltit, Jose M.; Franzini-Armstrong, Clara; Perez, Claudio F.

    2014-01-01

    The β1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca2+ release channel (RyR1). Progressive truncation of the β1a C terminus showed that deletion of amino acid residues Gln489 to Trp503 resulted in a loss of depolarization-induced Ca2+ release, a severe reduction of L-type Ca2+ currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu496, Leu500, and Trp503, which are thought to mediate direct β1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence 489QVQVLTSLRRNLSFW503 of β1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu496-Leu500-Trp503 within the β1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling. PMID:25384984

  9. Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris.

    PubMed

    Ostendorp, Thorsten; Weibel, Mirjam; Leclerc, Estelle; Kleinert, Peter; Kroneck, Peter M H; Heizmann, Claus W; Fritz, Günter

    2006-08-18

    RAGE is a multi-ligand receptor involved in various human diseases including diabetes, cancer or Alzheimer's disease. Engagement of RAGE by its ligands triggers activation of key cellular signalling pathways such as the MAP kinase and NF-kappaB pathways. Whereas the main isoform of RAGE is a transmembrane receptor with both extra- and intracellular domains, a secreted soluble isoform (sRAGE), corresponding to the extracellular part only, has the ability to block RAGE signalling and suppress cellular activation. Administration of sRAGE to animal models of cancer or multiple sclerosis blocked successfully tumour growth and the course of the autoimmune disease. These findings demonstrate that sRAGE may have a potential as therapeutic. We present here a fast and simple purification protocol of sRAGE from the yeast Pichia pastoris. The identity of the protein was confirmed by mass spectrometry and Western blot. The protein was N-glycosylated and 95-98% pure as judged by SDS-PAGE. PMID:16806067

  10. Melatonin inhibits insulin secretion in rat insulinoma β-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2.

    PubMed

    Mühlbauer, Eckhard; Albrecht, Elke; Hofmann, Kathleen; Bazwinsky-Wutschke, Ivonne; Peschke, Elmar

    2011-10-01

    Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic β-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma β-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease. PMID:21585522

  11. Urocortins and CRF type 2 receptor isoforms expression in the rat stomach are regulated by endotoxin: role in the modulation of delayed gastric emptying.

    PubMed

    Yuan, Pu-Qing; Wu, S Vincent; Taché, Yvette

    2012-07-01

    Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 μg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 μg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia. PMID:22517775

  12. Implication of cytochrome P-450 1A isoforms and the AH receptor in the genotoxicity of coal-tar fume condensate and bitumen fume condensates.

    PubMed

    Genevois, C; Pfohl-Leszkowicz, A; Boillot, K; Brandt, H; Castegnaro, M

    1998-06-01

    During the hot application of bitumen- or coal-tar-containing materials, fumes are emitted that contain polycyclic aromatic compounds. Although workers' exposure to these fumes is low, it might lead to health problems. No study has reported the metabolic pathways involved in the genotoxicity of coal tar or bitumen fume condensates (CTFC, BFCs). We have therefore studied the DNA adducts formed by incubation of CTFC or BFCs with liver microsomes from several type of mice and with yeast microsomes expressing individual human CYP enzymes. Our results demonstrates that: (1) the aryl hydrocarbon receptor (AHR) plays an important role in the biotransformation of BFCs and to a lesser extent of CTFC; (2) for CTFC, both cytochrome P450 (CYP) 1A isoforms are involved in the formation of genotoxic compounds, and the reactive metabolites formed via CYP 1A1, are substrates for epoxide hydrolase (mEH); (3) for BFCs, the genotoxicity is partially dependent upon CYP 1A1 and the reactive metabolites are not substrates for mEH; (4) CYP 1A isoforms are not exclusively responsible for the genotoxicity of the CTFC and BFCs as other CYPs and also enzymes of the [AH] gene battery, may play an important role. PMID:21781875

  13. The Paired Basic Amino Acid-cleaving Enzyme 4 (PACE4) Is Involved in the Maturation of Insulin Receptor Isoform B

    PubMed Central

    Kara, Imène; Poggi, Marjorie; Bonardo, Bernadette; Govers, Roland; Landrier, Jean-François; Tian, Sun; Leibiger, Ingo; Day, Robert; Creemers, John W. M.; Peiretti, Franck

    2015-01-01

    Gaining the full activity of the insulin receptor (IR) requires the proteolytic cleavage of its proform by intra-Golgi furin-like activity. In mammalian cells, IR is expressed as two isoforms (IRB and IRA) that are responsible for insulin action. However, only IRA transmits the growth-promoting and mitogenic effects of insulin-like growth factor 2. Here we demonstrate that the two IR isoforms are similarly cleaved by furin, but when this furin-dependent maturation is inefficient, IR proforms move to the cell surface where the proprotein convertase PACE4 selectively supports IRB maturation. Therefore, in situations of impaired furin activity, the proteolytic maturation of IRB is greater than that of IRA, and accordingly, the amount of phosphorylated IRB is also greater than that of IRA. We highlight the ability of a particular proprotein convertase inhibitor to effectively reduce the maturation of IRA and its associated mitogenic signaling without altering the signals emanating from IRB. In conclusion, the selective PACE4-dependent maturation of IRB occurs when furin activity is reduced; accordingly, the pharmacological inhibition of furin reduces IRA maturation and its mitogenic potential without altering the insulin effects. PMID:25527501

  14. Distinct roles of isoforms of the heme-liganded nuclear receptor E75, an insect orthologue of the vertebrate Rev-Erb, in mosquito reproduction

    PubMed Central

    Cruz, Josefa; Mane-Padros, Daniel; Zou, Zhen; Raikhel, Alexander S.

    2012-01-01

    Mosquitoes are adapted to using vertebrate blood as a nutrient source to promote egg development and as a consequence serve as disease vectors. Blood-meal activated reproductive events in female mosquitoes are hormonally and nutritionally controlled with an insect steroid hormone 20-hydroxyecdysone (20E) playing a central role. The nuclear receptor E75 is an essential factor in the 20E genetic hierarchy, however functions of its three isoforms - E75A, E75B, and E75C – in mosquito reproduction are unclear. By means of specific RNA interference depletion of E75 isoforms, we identified their distinct roles in regulating the level and timing of expression of key genes involved in vitellogenesis in the fat body (an insect analogue of vertebrate liver and adipose tissue) of the mosquito Aedes aegypti. Heme is required in a high level of expression of 20E-controlled genes in the fat body, and this heme action depends on E75. Thus, in mosquitoes, heme is an important signaling molecule, serving as a sensor of the availability of a protein meal for egg development. Disruption of this signaling pathway could be explored in the design of mosquito control approaches. PMID:22115961

  15. Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

    PubMed Central

    Xu, L; Lai, F A; Cohn, A; Etter, E; Guerrero, A; Fay, F S; Meissner, G

    1994-01-01

    Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels. Images PMID:8159742

  16. Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer

    PubMed Central

    Chellappa, Karthikeyani; Deol, Poonamjot; Evans, Jane R; Vuong, Linh M; Chen, Gang; Briançon, Nadege; Bolotin, Eugene; Lytle, Christian; Nair, Meera G; Sladek, Frances M

    2016-01-01

    HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb-/- mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer. DOI: http://dx.doi.org/10.7554/eLife.10903.001 PMID:27166517

  17. Opposing roles of nuclear receptor HNF4α isoforms in colitis and colitis-associated colon cancer.

    PubMed

    Chellappa, Karthikeyani; Deol, Poonamjot; Evans, Jane R; Vuong, Linh M; Chen, Gang; Briançon, Nadege; Bolotin, Eugene; Lytle, Christian; Nair, Meera G; Sladek, Frances M

    2016-01-01

    HNF4α has been implicated in colitis and colon cancer in humans but the role of the different HNF4α isoforms expressed from the two different promoters (P1 and P2) active in the colon is not clear. Here, we show that P1-HNF4α is expressed primarily in the differentiated compartment of the mouse colonic crypt and P2-HNF4α in the proliferative compartment. Exon swap mice that express only P1- or only P2-HNF4α have different colonic gene expression profiles, interacting proteins, cellular migration, ion transport and epithelial barrier function. The mice also exhibit altered susceptibilities to experimental colitis (DSS) and colitis-associated colon cancer (AOM+DSS). When P2-HNF4α-only mice (which have elevated levels of the cytokine resistin-like β, RELMβ, and are extremely sensitive to DSS) are crossed with Retnlb(-/-) mice, they are rescued from mortality. Furthermore, P2-HNF4α binds and preferentially activates the RELMβ promoter. In summary, HNF4α isoforms perform non-redundant functions in the colon under conditions of stress, underscoring the importance of tracking them both in colitis and colon cancer. PMID:27166517

  18. Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

    PubMed Central

    Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Puebla, Carlos; Guzmán-Gutiérrez, Enrique; Cifuentes, Fredi; Leiva, Andrea; Casanello, Paola; Sobrevia, Luis

    2011-01-01

    OBJECTIVE To determine whether insulin reverses gestational diabetes mellitus (GDM)–reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs). RESEARCH DESIGN AND METHODS Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine1177 phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist). RESULTS Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO–dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME. CONCLUSIONS GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM. PMID:21515851

  19. Synapse-associated protein-97 isoform-specific regulation of surface AMPA receptors and synaptic function in cultured neurons.

    PubMed

    Rumbaugh, Gavin; Sia, Gek-Ming; Garner, Craig C; Huganir, Richard L

    2003-06-01

    Members of the synapse-associated protein-97 (SAP97) family of scaffold proteins have been implicated as central organizers of synaptic junctions to build macromolecular signaling complexes around specific postsynaptic neurotransmitter receptors. In this regard, SAP97 has been suggested to regulate the synaptic localization of glutamate receptor type 1 subunits of the AMPA-type glutamate receptors. To test this hypothesis directly, we assessed the effects of SAP97 overexpression on surface expression of synaptic AMPA receptors. We find that recombinant SAP97 not only becomes concentrated at synaptic junctions but also leads to an increase in synaptic AMPA receptors, spine enlargement, and an increase in miniature EPSC (mEPSC) frequency, indicating that SAP97 has both postsynaptic and presynaptic effects on synaptic transmission. Synaptic targeting of SAP97, increased surface AMPA receptors, and increased mEPSC frequency are dependent on the presence of specific alternatively spliced sequences in SAP97 that encode a protein 4.1 binding site. These results suggest that SAP97 can affect the synaptic recruitment of AMPA receptors and spine morphology and that these effects may be regulated by alternative splicing. PMID:12805297

  20. Ligand-Binding Affinity at the Insulin Receptor Isoform-A and Subsequent IR-A Tyrosine Phosphorylation Kinetics are Important Determinants of Mitogenic Biological Outcomes

    PubMed Central

    Rajapaksha, Harinda; Forbes, Briony E.

    2015-01-01

    The insulin receptor (IR) is a tyrosine kinase receptor that can mediate both metabolic and mitogenic biological actions. The IR isoform-A (IR-A) arises from alternative splicing of exon 11 and has different ligand binding and signaling properties compared to the IR isoform-B. The IR-A not only binds insulin but also insulin-like growth factor-II (IGF-II) with high affinity. IGF-II acting through the IR-A promotes cancer cell proliferation, survival, and migration by activating some unique signaling molecules compared to those activated by insulin. This observation led us to investigate whether the different IR-A signaling outcomes in response to IGF-II and insulin could be attributed to phosphorylation of a different subset of IR-A tyrosine residues or to the phosphorylation kinetics. We correlated IR-A phosphorylation to activation of molecules involved in mitogenic and metabolic signaling (MAPK and Akt) and receptor internalization rates (related to mitogenic signaling). We also extended this study to incorporate two ligands that are known to promote predominantly mitogenic [(His4, Tyr15, Thr49, Ile51) IGF-I, qIGF-I] or metabolic (S597 peptide) biological actions, to see if common mechanisms can be used to define mitogenic or metabolic signaling through the IR-A. The threefold lower mitogenic action of IGF-II compared to insulin was associated with a decreased potency in activation of Y960, Y1146, Y1150, Y1151, Y1316, and Y1322, in MAPK phosphorylation and in IR-A internalization. With the poorly mitogenic S597 peptide, it was a decreased rate of tyrosine phosphorylation rather than potency that was associated with a low mitogenic potential. We conclude that both decreased affinity of IR-A binding and kinetics of IR-A phosphorylation can independently lead to a lower mitogenic activity. None of the studied parameters could account for the lower metabolic activity of qIGF-I. PMID:26217307

  1. Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.

    PubMed

    Patel, Seema; Mathonet, Pascale; Jaulent, Agnes M; Ullman, Christopher G

    2013-04-01

    WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications. PMID:23378640

  2. Molecular characterization and tissue distribution of aryl hydrocarbon receptor nuclear translocator isoforms, ARNT1 and ARNT2, and identification of novel splice variants in common cormorant (Phalacrocorax carbo).

    PubMed

    Lee, Jin-Seon; Kim, Eun-Young; Iwata, Hisato; Tanabe, Shinsuke

    2007-04-01

    High levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are accumulated in fish-eating birds including common cormorant (Phalacrocorax carbo). Most of the biochemical and toxic effects of TCDD are mediated by a basic helix-loop-helix and a conserved region among Per, ARNT, and Sim (bHLH/PAS) proteins, aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT). To study the molecular mechanism of TCDD toxicity in common cormorant as an avian model species, characterization of the AHR/ARNT signaling pathway in this species is necessary. The present study focuses on molecular characterization of ARNT from common cormorant (ccARNT). The cDNA of the ccARNT isoform, ccARNT1 obtained by the screening of hepatic cDNA library contains a 2424-bp open reading frame that encodes 807 amino acids, exhibiting high identities (92%) with chicken ARNT. This isoform contains a unique 22 amino acid residue in 3' end of PAS A domain as is also recognized in chicken ARNT. The ccARNT2 cDNA isolated from brain tissue has a 2151-bp open reading frame. The deduced amino acid sequence of ccARNT2 protein (716 aa) shows a conservation of bHLH and PAS motif in its N-terminal region with high similarities (96% and 78%, respectively) to that of ccARNT1. Using quantitative RT-PCR methods, the tissue distribution profiles of ccARNT1 and ccARNT2 were unveiled. Both ccARNT1 and ccARNT2 mRNAs were ubiquitously expressed in all examined tissues including liver. The expression profile of ccARNT1 was comparable with that of rodent ARNT1, but ccARNT2 was not with rodent ARNT2, implying different roles of ARNT2 between the two species. There was a significant positive correlation between ARNT1 and ARNT2 mRNA expression levels in the liver of wild cormorant population, indicating that their expressions may be enforced by similar transcriptional regulation mechanism. Novel variants of ccARNT1 and ccARNT2 isoforms that were supposed to

  3. Delayed Parturition and Altered Myometrial Progesterone Receptor Isoform A Expression in Mice Null for Kruppel-like Factor 9

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pre-term and delayed labor conditions are devastating health problems, with currently unknown etiologies. We previously showed that the transcription factor Krüppel-like factor 9 (KLF9) influences the expression and/or transcriptional activity of receptors for estrogen and progesterone in endometria...

  4. Muscle-specific mRNA isoform encodes a protein composed mainly of the N-terminal 175 residues of type 2 Ins(1,4,5)P3 receptor.

    PubMed Central

    Futatsugi, A; Kuwajima, G; Mikoshiba, K

    1998-01-01

    We have found a novel isoform of the mouse type 2 Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] mRNA by reverse transcriptase-mediated PCR analysis. The novel isoform, which was expressed specifically in skeletal muscle and heart, was generated by the inclusion of a novel exon. As this exon contains a stop codon, the isoform encodes a putative protein (designated TIPR) consisting of 175 acid residues of the type 2 Ins(1,4,5)P3R and the following six residues derived from this exon. We transfected the cDNA of this isoform into COS-7 cells; these cells expressed a 24 kDa protein that was recognized by an antibody against TIPR produced in Escherichia coli. The isoform encoding TIPR was also found in human skeletal muscle and heart. The N-terminal region of Ins(1,4,5)P3R is suggested to have a role in ligand binding and to interact with the C-terminal channel domain of Ins(1,4,5)P3R itself. TIPR might regulate the Ins(1,4,5)P3 signal pathway in both muscles. PMID:9729462

  5. Sequence Analysis and Identification of New Isoform of EP4 Receptors in Different Atlantic Salmon Tissues (Salmo salar L.) and Its Role in PGE2 Induced Immunomodulation In Vitro

    PubMed Central

    Koenig, Melanie; Evensen, Øystein

    2015-01-01

    PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5’ untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1β genes expression in a time dependent manner and without cAMP upregulation. PMID:25837516

  6. Asymmetric Total Synthesis of (+)-Ryanodol and (+)-Ryanodine.

    PubMed

    Masuda, Kengo; Koshimizu, Masaki; Nagatomo, Masanori; Inoue, Masayuki

    2016-01-01

    (+)-Ryanodine (1) is the ester derivative of 1H-pyrrole-2-carboxylic acid and the complex terpenoid (+)-ryanodol (2), which possesses eleven contiguous stereogenic centers on the ABCDE-ring system. Compound 1 is known to be a potent modulator of intracellular calcium release channels, whereas the activity of 2 is significantly weaker. To chemically construct 1, the multiple oxygen functional groups must be installed on the fused pentacycle in stereoselective fashions and the extremely hindered C3-hydroxy group must be acylated in a site-selective manner. First, the total synthesis of 2 was accomplished by introducing the five stereocenters from the previously prepared enantiopure ABDE-ring 7. Stereoselective construction of the C3-secondary, C2- and C6-tertiary alcohols was achieved by three nucleophilic reactions. The C9- and C10-trisubstituted carbon centers were regio- and stereoselectively introduced by hydroboration/oxidation of the six-membered C-ring, which was formed by the ring-closing metathesis reaction. Direct esterification of the C3-alcohol with pyrrole-2-carboxylic acid proved unsuccessful; therefore, we developed a new, two-step protocol for attachment of the pyrrole moiety. The C3-hydroxy group was first converted into the less sterically cumbersome glycine ester, which was then transformed into the pyrrole ring through condensation with 1,3-bis(dimethylamino)allylium tetrafluoroborate. This procedure resulted in the first total synthesis of 1. PMID:26616265

  7. Differential expression of olfactory genes in the southern house mosquito and insights into unique odorant receptor gene isoforms

    PubMed Central

    Leal, Walter S.; Choo, Young-Moo; Xu, Pingxi; da Silva, Cherre S. B.; Ueira-Vieira, Carlos

    2013-01-01

    The southern house mosquito, Culex quinquefasciatus, has one of the most acute and eclectic olfactory systems of all mosquito species hitherto studied. Here, we used Illumina sequencing to identify olfactory genes expressed predominantly in antenna, mosquito’s main olfactory organ. Less than 50% of the trimmed reads generated by high-quality libraries aligned to a transcript, but approximately 70% of them aligned to the genome. Differential expression analysis, which was validated by quantitative real-time PCR on a subset of genes, showed that approximately half of the 48 odorant-binding protein genes were enriched in antennae, with the other half being predominantly expressed in legs. Similar patterns were observed with chemosensory proteins, “plus-C” odorant-binding proteins, and sensory neuron membrane proteins. Transcripts for as many as 43 ionotropic receptors were enriched in female antennae, thus making the ionotropic receptor family the largest of antennae-rich olfactory genes, second only to odorant receptor (OR) genes. As many as 177 OR genes have been identified, including 36 unique transcripts. The unique OR genes differed from previously annotated ORs in internal sequences, splice variants, and extended N or C terminus. One of the previously unknown transcripts was validated by cloning and functional expression. When challenged with a large panel of physiologically relevant compounds, CquiOR95b responded in a dose-dependent manner to ethyl 2-phenylacteate, which was demonstrated to repel Culex mosquitoes, and secondarily to citronellal, a known insect repellent. This transcriptome study led to identification of key molecular components and a repellent for the southern house mosquito. PMID:24167245

  8. Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

    PubMed Central

    Nelander, Gitte-Mai; Hansen, Bo Falck; Jensen, Pia; Krabbe, Jonas S.; Jensen, Marianne B.; Hegelund, Anne Charlotte; Svendsen, Jette E.; Oleksiewicz, Martin B.

    2009-01-01

    Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing. PMID:19898946

  9. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    PubMed Central

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  10. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  11. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors.

    PubMed

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O; Bychkov, M L; Paramonov, A S; Chugunov, A O; Kulbatskii, D S; Arvaniti, M; Dolejsi, Eva; Schaer, T; Arseniev, A S; Efremov, R G; Thomsen, M S; Dolezal, V; Bertrand, D; Dolgikh, D A; Kirpichnikov, M P

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  12. NF-κB and Androgen Receptor Variant 7 Induce Expression of SRD5A Isoforms and Confer 5ARI Resistance

    PubMed Central

    Austin, David C.; Strand, Douglas W.; Love, Harold L.; Franco, Omar E.; Grabowska, Magdalena M.; Miller, Nicole L.; Hameed, Omar; Clark, Peter E.; Matusik, Robert J.; Jin, Ren J.; Hayward, Simon W.

    2016-01-01

    BACKGROUND Benign prostatic hyperplasia (BPH) is treated with 5α-reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF-κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy. METHODS Tissue was collected from “Surgical” patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from “Incidental” patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression. RESULTS SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions. CONCLUSION Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism

  13. Characterization of Novel Peroxisome Proliferator-activated Receptor γ Coactivator-1α (PGC-1α) Isoform in Human Liver*

    PubMed Central

    Felder, Thomas K.; Soyal, Selma M.; Oberkofler, Hannes; Hahne, Penelope; Auer, Simon; Weiss, Richard; Gadermaier, Gabriele; Miller, Karl; Krempler, Franz; Esterbauer, Harald; Patsch, Wolfgang

    2011-01-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator that contributes to the regulation of numerous transcriptional programs including the hepatic response to fasting. Mechanisms at transcriptional and post-transcriptional levels allow PGC-1α to support distinct biological pathways. Here we describe a novel human liver-specific PGC-1α transcript that results from alternative promoter usage and is induced by FOXO1 as well as glucocorticoids and cAMP-response element-binding protein signaling but is not present in other mammals. Hepatic tissue levels of novel and wild-type transcripts were similar but were only moderately associated (p < 0.003). Novel mRNA levels were associated with a polymorphism located in its promoter region, whereas wild-type transcript levels were not. Furthermore, hepatic PCK1 mRNA levels exhibited stronger associations with the novel than with the wild-type transcript levels. Except for a deletion of 127 amino acids at the N terminus, the protein, termed L-PGC-1α, is identical to PGC-1α. L-PGC-1α was localized in the nucleus and showed coactivation properties that overlap with those of PGC-1α. Collectively, our data support a role of L-PGC-1α in gluconeogenesis, but functional differences predicted from the altered structure suggest that L-PGC-1α may have arisen to adapt PGC-1α to more complex metabolic pathways in humans. PMID:22009745

  14. Differential Roles of PML Isoforms.

    PubMed

    Nisole, Sébastien; Maroui, Mohamed Ali; Mascle, Xavier H; Aubry, Muriel; Chelbi-Alix, Mounira K

    2013-01-01

    The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed. PMID:23734343

  15. Promoter hypermethylation of progesterone receptor isoform B (PR-B) in adenomyosis and its rectification by a histone deacetylase inhibitor and a demethylation agent.

    PubMed

    Jichan Nie; Xishi Liu; Guo, Sun-Wei

    2010-11-01

    Adenomyosis is a fairly common gynecologic disease with unknown pathogenesis. We sought to investigate as to whether the promoter of progesterone receptor isoform B (PR-B) is hypermethylated in adenomyosis and to investigate the treatment of ectopic endometrial stromal cells with trichostatin A (TSA), a histone deacetylase inhibitor (HDI), and 5-aza-2-deoxycytidine (ADC), a demethylation agent, on PR-B gene and protein expression, and on cell viability. Ectopic endometrial tissue specimens were obtained from 9 women with adenomyosis whereas control endometrial tissue samples were obtained from 8 women with surgically diagnosed benign ovarian cysts but without any clinical history of endometriosis/adenomyosis/ myoma. Endometrial stromal cells were isolated, purified, cultured, and analyzed by methylation-specific polymerase chain reaction (PCR), real-time reverse transcriptase PCR (RT-PCR), and Western blot analysis, cell viability assays, and fluorescence-activated cell sorting. We found that none of the normal endometrial stromal cells had PR-B promoter methylation. In contrast, 2 out of 3 ectopic endometrial stromall cells had PR-B hypermethylation (P < .05). The treatment with both TSA and ADC elevated PR-B gene and protein expression in ectopic, but not in normal, endometrial stromal cells. Both TSA and ADC treatment dose-dependently reduced cell viability of ectopic endometrial stromal cells. Trichostatin A and ADC treatment also suppressed the cell cycle progression in ectopic endometrial stromal cells. Thus, this study provides the first piece of evidence that adenomyosis has epigenetic aberration and may also be an epigenetic disease amenable to rectification by pharmacological means. This perspective may shed new light onto the pathogenesis of adenomyosis and lead to novel ways to treat the disease. PMID:20697142

  16. Expression of autocrine prolactin and the short isoform of prolactin receptor are associated with inflammatory response and apoptosis in monocytes stimulated with Mycobacterium bovis proteins.

    PubMed

    López-Rincón, Gonzalo; Mancilla, Raúl; Pereira-Suárez, Ana L; Martínez-Neri, Priscila A; Ochoa-Zarzosa, Alejandra; Muñoz-Valle, José Francisco; Estrada-Chávez, Ciro

    2015-06-01

    Increased levels of prolactin (PRL) have recently been associated with carcinogenesis and the exacerbation of autoimmune diseases, and might be involved in the progression of tuberculosis (TB). To investigate the relationship between PRL and prolactin receptor (PRLr) expression with inflammatory response and apoptosis in monocytes, we used THP-1 cells stimulated with antigens of the Mycobacterium bovis AN5 strain culture filtrate protein (CFP-M. bovis). Western blot (WB), real-time Polymerase chain reaction (PCR), and immunocytochemistry were performed to identify both PRL and PRLr molecules. PRL bioactivity and proinflammatory cytokine detection were assessed. The results showed that PRL and PRLr messenger RNA (mRNA) were synthesized in THP-1 monocytes induced with CFP-M. bovis at peaks of 176- and 404-fold, respectively. PRL forms of 60 and 80kDa and PRLr isoforms of 40, 50, and 65kDa were also identified as time-dependent, while 60-kDa PRL, as well as 40-, and 50-kDa PRLr, were found as soluble forms in culture media and later in the nucleus of THP-1 monocytes. PRL of 60kDa released by monocytes exhibited bioactivity in Nb2 cells, and both synthesized PRL and synthesized PRLr were related with nitrite and proinflammatory cytokine levels proapoptotic activity in CFP-M. bovis-induced monocytes. Our results suggest the overexpression of a full-autocrine loop of PRL and PRLr in monocytes that enhances the inflammatory response and apoptosis after priming with M. bovis antigens. PMID:25797370

  17. Developmental toxicity of 4-ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH receptor isoforms and hepatic cytochrome P4501A metabolism

    SciTech Connect

    Incardona, John P. . E-mail: john.incardona@noaa.gov; Day, Heather L.; Collier, Tracy K.; Scholz, Nathaniel L.

    2006-12-15

    Polycyclic aromatic hydrocarbons (PAHs) derived from fossil fuels are ubiquitous contaminants and occur in aquatic habitats as highly variable and complex mixtures of compounds containing 2 to 6 rings. For aquatic species, PAHs are generally accepted as acting through either of two modes of action: (1) 'dioxin-like' toxicity mediated by activation of the aryl hydrocarbon receptor (AHR), which controls a battery of genes involved in PAH metabolism, such as cytochrome P4501A (CYP1A) and (2) 'nonpolar narcosis', in which tissue uptake is dependent solely on hydrophobicity and toxicity is mediated through non-specific partitioning into lipid bilayers. As part of a systematic analysis of mechanisms of PAH developmental toxicity in zebrafish, we show here that three tetracyclic PAHs (pyrene, chrysene, and benz[a]anthracene) activate the AHR pathway tissue-specifically to induce distinct patterns of CYP1A expression. Using morpholino knockdown of ahr1a, ahr2, and cyp1a, we show that distinct embryolarval syndromes induced by exposure to two of these compounds are differentially dependent on tissue-specific activation of AHR isoforms or metabolism by CYP1A. Exposure of embryos with and without circulation (silent heart morphants) resulted in dramatically different patterns of CYP1A induction, with circulation required to deliver some compounds to internal tissues. Therefore, biological effects of PAHs cannot be predicted simply by quantitative measures of AHR activity or a compound's hydrophobicity. These results indicate that current models of PAH toxicity in fish are greatly oversimplified and that individual PAHs are pharmacologically active compounds with distinct and specific cellular targets.

  18. Type I (RI) and type II (RII) receptors for transforming growth factor-beta isoforms are expressed subsequent to transforming growth factor-beta ligands during excisional wound repair.

    PubMed Central

    Gold, L. I.; Sung, J. J.; Siebert, J. W.; Longaker, M. T.

    1997-01-01

    Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only

  19. Mitochondrial Ryanodine Receptors and Other Mitochondrial Ca2+ Permeable Channels

    PubMed Central

    Ryu, Shin-Young; Beutner, Gisela; Dirksen, Robert T.; Kinnally, Kathleen W.; Sheu, Shey-Shing

    2010-01-01

    Ca2+ channels that underlie mitochondrial Ca2+ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca2+ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca2+ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca2+ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca2+ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca2+ channels. PMID:20096690

  20. Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development

    SciTech Connect

    Incardona, John P. Linbo, Tiffany L.; Scholz, Nathaniel L.

    2011-12-15

    Petroleum-derived compounds, including polycyclic aromatic hydrocarbons (PAHs), commonly occur as complex mixtures in the environment. Recent studies using the zebrafish experimental model have shown that PAHs are toxic to the embryonic cardiovascular system, and that the severity and nature of this developmental cardiotoxicity varies by individual PAH. In the present study we characterize the toxicity of the relatively higher molecular weight 5-ring PAHs benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), and benzo[k]fluoranthene (BkF). While all three compounds target the cardiovascular system, the underlying role of the ligand-activated aryl hydrocarbon receptor (AHR2) and the tissue-specific induction of the cytochrome p450 metabolic pathway (CYP1A) were distinct for each. BaP exposure (40 {mu}M) produced AHR2-dependent bradycardia, pericardial edema, and myocardial CYP1A immunofluorescence. By contrast, BkF exposure (4-40 {mu}M) caused more severe pericardial edema, looping defects, and erythrocyte regurgitation through the atrioventricular valve that were AHR2-independent (i.e., absent myocardial or endocardial CYP1A induction). Lastly, exposure to BeP (40 {mu}M) yielded a low level of CYP1A+ signal in the vascular endothelium of the head and trunk, without evident toxic effects on cardiac function or morphogenesis. Combined with earlier work on 3- and 4-ring PAHs, our findings provide a more complete picture of how individual PAHs may drive the cardiotoxicity of mixtures in which they predominate. This will improve toxic injury assessments and risk assessments for wild fish populations that spawn in habitats altered by overlapping petroleum-related human impacts such as oil spills, urban stormwater runoff, or sediments contaminated by legacy industrial activities. -- Highlights: Black-Right-Pointing-Pointer PAH compounds with 5 rings in different arrangements caused differential tissue-specific patterns of CYP1A induction in zebrafish embryos. Black

  1. Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: identification, molecular cloning and effects on cell migration.

    PubMed Central

    Zannettino, Andrew C W; Roubelakis, Maria; Welldon, Katie J; Jackson, Denise E; Simmons, Paul J; Bendall, Linda J; Henniker, Anthony; Harrison, Kate L; Niutta, Silvana; Bradstock, Kenneth F; Watt, Suzanne M

    2003-01-01

    SHP-2 (Src homology phosphatase type-2) is essential for haematopoietic skeletal and vascular development. Thus the identification of its binding partners is critically important. In the present study, we describe a unique monoclonal antibody, WM78, which interacts with PZR, a SHP-2 binding partner. Furthermore, we identify two novel isoforms of PZR, PZRa and PZRb, derived by differential splicing from a single gene transcription unit on human chromosome 1q24. All are type 1 transmembrane glycoproteins with identical extracellular and transmembrane domains, but differ in their cytoplasmic tails. The PZR intracellular domain contains two SHP-2 binding immunoreceptor tyrosine-based inhibitory motifs (VIY(246)AQL and VVY(263)ADI) which are not present in PZRa and PZRb. Using the WM78 monoclonal antibody, which recognizes the common extracellular domain of the PZR isoforms, we demonstrate that the PZR molecules are expressed on mesenchymal and haematopoietic cells, being present on the majority of CD34(+)CD38(+) and early clonogenic progenitors, and at lower levels on CD34(+)CD38(-) cells and the hierarchically more primitive pre-colony forming units. Interestingly, we show by reverse transcriptase-PCR that the PZR isoforms are differentially expressed in haematopoietic, endothelial and mesenchymal cells. Both PZR and PZRb are present in CD133(+) precursors and endothelial cells, PZRb predominates in mesenchymal and committed myelomonocytic progenitor cells, and all three isoforms occur in erythroid precursor cell lines. Importantly, using SHP-2 mutant (Delta 46-110) and SHP-2 rescue of embryonic fibroblasts stably expressing the PZR isoforms, we demonstrate for the first time that PZR, but not PZRa or PZRb, facilitates fibronectin- dependent migration of cells expressing a competent SHP-2 molecule. These observations will be instrumental in determining the mechanisms whereby PZR isoforms regulate cell motility. PMID:12410637

  2. Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

    PubMed

    Yousaf, N; Low, W Y; Onipinla, A; Mein, C; Caulfield, M; Munroe, P B; Chernajovsky, Y

    2015-05-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme. PMID:25545008

  3. Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

    SciTech Connect

    Gerthoffer, W.T.; Murphey, K.A.; Khoyi, M.A.

    1988-08-01

    Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased /sup 45/Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on /sup 45/Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.

  4. ISOFORM-SPECIFIC BINDING OF 14-3-3 PROTEINS TO NITRATE REDUCTASE AND THE BRASSINOSTEROID INSENSITIVE 1 RECEPTOR KINASE SIGNALING COMPLEX

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are known to bind many different soluble protein clients, but less is known about binding to integral membrane proteins, and in both cases the issue of isoform specificity remains largely unexplored. Using an array of anti-14-3-3 antibodies and 2-dimensional electrophoresis (2-DE...

  5. Differential effects of retinoic acid on the growth of isogenic metastatic and non-metastatic breast cancer cell lines and their association with distinct expression of retinoic acid receptor beta isoforms 2 and 4.

    PubMed

    Hayashi, Ken; Goodison, Steven; Urquidi, Virginia; Tarin, David; Lotan, Reuben; Tahara, Eiichi

    2003-03-01

    The human retinoic acid receptor beta (RARbeta) has three isoforms (beta1, beta2, and beta4), which play important, distinct roles in mediating the effects of retinoic acid on cell growth and apoptosis. Whereas RARbeta2 is a potent inhibitor of breast cancer cell proliferation, RARbeta4 can act as a dominant-negative repressor of RARbeta2-mediated growth suppression. In this study we investigated the effects of all-trans-retinoic acid (ATRA) on two clones derived from the breast cancer cell line MDA-MB-435: a non-metastatic clone (NM-2C5) and a metastatic clone (M-4A4). ATRA treatment of the NM-2C5 cells resulted in growth inhibition and apoptosis, whereas the M-4A4 cells were resistant to ATRA. Analyses of the expression of RARbeta isoforms revealed that the sensitive NM-2C5 clone expressed only RARbeta2, whereas the resistant M-4A4 cells expressed both RARbeta2 and RARbeta4 mRNA and protein. ATRA treatment increased RARbeta2 mRNA level in NM-2C5 cells, whereas the same treatment of the M-4A4 cells resulted in an increase in RARbeta4 and a decrease in RARbeta2 mRNA. ATRA treatment of NM-2C5 cells increased the protein levels of the histone acetyl transferases p300 and CBP, suppressed the level of histone deacetylase and increased the level of acetylated histone H4. ATRA also decreased Bcl-2 and increased Bax and decreased VEGF. In contrast, the same treatment of the M-4A4 cells resulted in opposite effects. These results suggest that the effects of ATRA on the growth of the metastatic and non-metastatic breast cancer cell lines depend on the expression of RARbeta isoforms and that the expression of RARbeta4 may contribute to metastatic properties. PMID:12579317

  6. Tropomyosin isoforms and reagents

    PubMed Central

    Schevzov, Galina; Whittaker, Shane P; Fath, Thomas; Lin, Jim JC

    2011-01-01

    Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments.1 The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike. PMID:22069507

  7. Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms.

    PubMed Central

    Vanlingen, S; Sipma, H; De Smet, P; Callewaert, G; Missiaen, L; De Smedt, H; Parys, J B

    2000-01-01

    We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP(3)R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP(3)-binding domain and bound IP(3) and adenophostin A with high affinity. Ca(2+) and calmodulin were previously found to maximally inhibit IP(3) binding to lbs-1 by 42+/-6 and 43+/-6% respectively, and with an IC(50) of approx. 200 nM and 3 microM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca(2+) inhibited IP(3) binding to lbs-3 with an IC(50) of approx. 700 nM (37+/-4% inhibition at 5 microM Ca(2+)), while IP(3) binding to lbs-2 was not affected by increasing [Ca(2+)] from 100 nM to 25 microM. Calmodulin (10 microM) inhibited IP(3) binding to lbs-3 by 37+/-4%, while IP(3) binding to lbs-2 was inhibited by only 11+/-2%. The inhibition of IP(3) binding to lbs-3 by calmodulin was dose-dependent (IC(50) approximately 2 microM). We conclude that the IP(3)-binding domains of the various IP(3)R isoforms differ in binding characteristics for IP(3) and adenophostin A, and are differentially modulated by Ca(2+) and calmodulin, suggesting that the various IP(3)R isoforms can have different intracellular functions. PMID:10677344

  8. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  9. Apolipoprotein E isoform-dependent microglia migration

    PubMed Central

    Cudaback, Eiron; Li, Xianwu; Montine, Kathleen S.; Montine, Thomas J.; Keene, C. Dirk

    2011-01-01

    Complement component C5a and ATP are potent effectors of microglial movement and are increased in diverse neurodegenerative diseases and at sites of injury. Apolipoprotein E (apoE) influences microglial function, and different human apoE isoforms confer variable risk for development of neurodegenerative disorders, especially Alzheimer's disease. The purpose of this investigation was to test the hypothesis that mouse apoE and human apoE isoforms influence microglial migration. Using primary wild-type and apoE-deficient microglia, we show that C5a- and ATP-stimulated chemotaxis are largely apoE-dependent processes with different molecular bases. Although the C5a-dependent chemotaxis of wild-type microglia was completely blocked by receptor-associated protein (RAP), suggesting apoE receptor involvement, ATP-stimulated migration was unaffected by RAP but was associated with differential ERK phosphorylation. Studies using primary microglia derived from targeted replacement mice “humanized” for the coding exons (protein isoform) of human ε2 (apoE2), ε3 (apoE3), or ε4 (apoE4) allele of APOE revealed that primary mouse microglia expressing apoE4 or apoE2 exhibited significantly reduced C5a- and ATP-stimulated migration compared with microglia expressing human apoE3. This study, for the first time, demonstrates apoE dependence and apoE isoform-specific modulation of microglial migration in response to distinct chemotactic stimuli commonly associated with neurodegenerative disease.—Cudaback, E., Li, X., Montine, K. S., Montine, T. J., Keene, C. D. Apolipoprotein E isoform-dependent microglia migration. PMID:21385991

  10. A novel substituted aminoquinoline selectively targets voltage-sensitive sodium channel isoforms and NMDA receptor subtypes and alleviates chronic inflammatory and neuropathic pain.

    PubMed

    Tabakoff, Boris; Ren, Wenhua; Vanderlinden, Lauren; Snell, Lawrence D; Matheson, Christopher J; Wang, Ze-Jun; Levinson, Rock; Smothers, C Thetford; Woodward, John J; Honse, Yumiko; Lovinger, David; Rush, Anthony M; Sather, William A; Gustafson, Daniel L; Hoffman, Paula L

    2016-08-01

    Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models. PMID:27158117

  11. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

    PubMed

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A; Di Pretoro, Simona; Pires, Susana S; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A; Hossbach, Markus; MacLaren, Robert E; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W; Wood, Matthew J A; Foster, Russell G; Peirson, Stuart N

    2015-09-21

    Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  12. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    PubMed Central

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  13. Effect of IP3 and ryanodine treatments on the development of bovine parthenogenetic and reconstructed embryos.

    PubMed

    Ahn, G J; Lee, B C; Hwang, W S

    2001-08-01

    For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos. PMID:14614284

  14. Mapping of the {beta}{sub 2} subunit gene (GABRB2) to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABA{sub A} receptor isoform

    SciTech Connect

    Russek, S.J.; Farb, D.H. |

    1994-10-01

    The {gamma}-aminobutyric acid receptor (GABA{sub A}R) is a multisubunit Cl{sup -} channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABA{sub A}R subunits, including {alpha}{sub 1-6}, {beta}{sub 1-4}, {gamma}{sub 1-4}, {sigma}, and {rho}{sub 1-2}, suggesting that an enormous number of combinations of subunits are possible. Here we report that the {beta}{sub 2} gene is located on chromosome 5q34-q35, defining a cluster comprising {alpha}{sub 1}, {beta}{sub 2}, and {gamma}{sub 2} genes that together code for the most abundant GABA{sub A}R isoform. The fact that intron position is conserved in the {beta}{sub 1-3} genes, taken together with the observation that chromosomes 4 and 15 also contain distinct {alpha}-{beta}-{gamma} gene clusters, strongly suggests that an ancestral {alpha}-{beta}-{gamma} cluster was duplicated and translocated to at least two different chromosomes. This organization of GABA{sub A}R gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression. 34 refs., 5 figs., 1 tab.

  15. Expression of gene, protein and immunohistochemical localization of the estrogen receptor isoform ERα1 in male rainbow trout lymphoid organs; indication of the role of estrogens in the regulation of immune mechanisms.

    PubMed

    Massart, Sophie; Milla, Sylvain; Kestemont, Patrick

    2014-08-01

    In vertebrates, estrogens act on the reproductive system but also affect the functioning of non-reproductive tissues such as the immune system. In teleost fish, effects of estrogens and xenoestrogens have been reported extensively, but the available information on targeted tissues and cells is still scarce. Moreover, a better knowledge of the distinct ER subtypes is required to find out the mechanistic pathways by which estrogen compounds are able to disrupt endogenous estrogen signaling in fish immunity. The present study aimed at characterizing, in male rainbow trout juveniles, multi-tissue gene expression pattern of one isoform of estrogen receptor (ER), ERα1, at the mRNA and protein levels. The mRNA levels for ERα1 were measured in various lymphoid organs by real-time RT-PCR and ERα1 protein level by Western blot. Furthermore, this protein was located by immunohistochemistry in the same organs. The transcripts were ubiquitously expressed, but at a higher level in testis and liver, while the protein was more abundant in testis and skin. Moreover, the ERα1 was detected in endothelial, Kupffer, mucous and chloride cells, hematopoietic tissues, proximal tubule, epithelia of the skin and intestine, in the lamina propria and in the stratum granulosum. This distribution backs the idea that, in male rainbow trout, estrogeno-mimetic compounds could be involved in different immune mechanisms such as inflammatory response, transport of Ig, mucus production, regulation of cellular immunity and development and maturation of lymphoid and myeloid cells. PMID:24937418

  16. Changes in Knee Laxity and Relaxin Receptor Isoforms Expression (RXFP1/RXFP2) in the Knee throughout Estrous Cycle Phases in Rodents

    PubMed Central

    Dehghan, Firouzeh; Soori, Rahman; Dehghan, Parvin; Gholami, Khadijeh; Muniandy, Sekaran; Azarbayjani, Mohammad Ali; Yusof, Ashril

    2016-01-01

    The changes in knee laxity and relaxin receptor expression at different phases of rodent estrous cycle are not known. Here, changes in the parameter were investigated in rats at different phases of the estrous cycle. Estrous cycle phases of intact female rats were determined by cytological examination of the vaginal smear. Following phase identification, blood was collected for serum hormone analyses. Knee passive range of motion (ROM) was determined by using a digital miniature goniometer. The animals were then sacrificed and patellar tendon, collateral ligaments and hamstring muscles were harvested for relaxin/insulin-like family peptide receptor 1 and 2 (RXFP1/RXFP2) analyses. Knee passive ROM was the highest at proestrus followed by diestrus and the lowest at estrus. Estrogen level was the highest at proestrus while progesterone and relaxin levels were the highest at diestrus. A strong correlation was observed between relaxin and progesterone levels. At proestrus, expression of RXFP1 and RXFP2 proteins and mRNAs were the highest at proestrus followed by diestrus and estrus. The finding shows that higher level of progesterone and relaxin in diestrus might be responsible for higher laxity of knee joint in rats. PMID:27513858

  17. Changes in Knee Laxity and Relaxin Receptor Isoforms Expression (RXFP1/RXFP2) in the Knee throughout Estrous Cycle Phases in Rodents.

    PubMed

    Dehghan, Firouzeh; Soori, Rahman; Dehghan, Parvin; Gholami, Khadijeh; Muniandy, Sekaran; Azarbayjani, Mohammad Ali; Yusof, Ashril

    2016-01-01

    The changes in knee laxity and relaxin receptor expression at different phases of rodent estrous cycle are not known. Here, changes in the parameter were investigated in rats at different phases of the estrous cycle. Estrous cycle phases of intact female rats were determined by cytological examination of the vaginal smear. Following phase identification, blood was collected for serum hormone analyses. Knee passive range of motion (ROM) was determined by using a digital miniature goniometer. The animals were then sacrificed and patellar tendon, collateral ligaments and hamstring muscles were harvested for relaxin/insulin-like family peptide receptor 1 and 2 (RXFP1/RXFP2) analyses. Knee passive ROM was the highest at proestrus followed by diestrus and the lowest at estrus. Estrogen level was the highest at proestrus while progesterone and relaxin levels were the highest at diestrus. A strong correlation was observed between relaxin and progesterone levels. At proestrus, expression of RXFP1 and RXFP2 proteins and mRNAs were the highest at proestrus followed by diestrus and estrus. The finding shows that higher level of progesterone and relaxin in diestrus might be responsible for higher laxity of knee joint in rats. PMID:27513858

  18. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  19. DNA signals at isoform promoters.

    PubMed

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  20. Expression of 15-Hydroxyprostaglandin Dehydrogenase in Human Chorion Is Associated with Peroxisome Proliferator-Activated Receptor Isoform Expression in Term Labor.

    PubMed

    He, Ping; Li, Yuan; Ding, Xiaoying; Sun, Qianqian; Huang, Ying; Gu, Hang; Ni, Xin

    2015-07-01

    Chorionic NAD-dependent 15-hydroxy prostaglandin dehydrogenase (PGDH) plays a pivotal role in controlling the amount of prostaglandins in the uterus. Peroxisome proliferator-activated receptors (PPARs) are implicated to be involved in parturition. In this study, we investigated whether PPARs are involved in control of PGDH expression in chorion. The chorionic tissues were collected from the following groups of the women with singleton pregnancy: term no labor (TNL), term labor (TL) and preterm labor (PTL). Chorionic trophoblasts were isolated and cultured in vitro. Immunocytochemistry analysis showed that PPARα, PPARβ, and PPARγ were localized to trophoblasts in chorion. The protein levels of PGDH, PPARβ, and PPARγ were localized to trophoblasts in chorion. The protein levels of PPARα, PPARβ, and PPARγ were reduced in TL tissues compared to that of TNL group. PPARα, PPARβ, and PPARγ expression correlated to PGDH in TNL tissues, whereas only PPARγ expression correlated to PGDH in TL chorion tissues. PGDH expression was decreased in PTL tissues compared with TL group, whereas the expression of PPARs was not significantly different between TL and PTL groups. The agonists of three PPARs dose-dependently stimulated PGDH activity, mRNA, and protein expression in cultured chorionic cells. PPARs did not affect the stability of PGDH mRNA but stimulated the transcriptional activity of HPGD gene. Our results suggest that PPARs play pivotal roles in maintenance of PGDH expression in chorion during human pregnancy. PMID:26093984

  1. Electrophysiological effects of ryanodine derivatives on the sheep cardiac sarcoplasmic reticulum calcium-release channel.

    PubMed Central

    Tinker, A; Sutko, J L; Ruest, L; Deslongchamps, P; Welch, W; Airey, J A; Gerzon, K; Bidasee, K R; Besch, H R; Williams, A J

    1996-01-01

    We have examined the effects of a number of derivatives of ryanodine on K+ conduction in the Ca2+ release channel purified from sheep cardiac sarcoplasmic reticulum (SR). In a fashion comparable to that of ryanodine, the addition of nanomolar to micromolar quantities to the cytoplasmic face (the exact amount depending on the derivative) causes the channel to enter a state of reduced conductance that has a high open probability. However, the amplitude of that reduced conductance state varies between the different derivatives. In symmetrical 210 mM K+, ryanodine leads to a conductance state with an amplitude of 56.8 +/- 0.5% of control, ryanodol leads to a level of 69.4 +/- 0.6%, ester A ryanodine modifies to one of 61.5 +/- 1.4%, 9,21-dehydroryanodine to one of 58.3 +/- 0.3%, 9 beta,21beta-epoxyryanodine to one of 56.8 +/- 0.8%, 9-hydroxy-21-azidoryanodine to one of 56.3 +/- 0.4%, 10-pyrroleryanodol to one of 52.2 +/- 1.0%, 3-epiryanodine to one of 42.9 +/- 0.7%, CBZ glycyl ryanodine to one of 29.4 +/- 1.0%, 21-p-nitrobenzoyl-amino-9-hydroxyryanodine to one of 26.1 +/- 0.5%, beta-alanyl ryanodine to one of 14.3 +/- 0.5%, and guanidino-propionyl ryanodine to one of 5.8 +/- 0.1% (chord conductance at +60 mV, +/- SEM). For the majority of the derivatives the effect is irreversible within the lifetime of a single-channel experiment (up to 1 h). However, for four of the derivatives, typified by ryanodol, the effect is reversible, with dwell times in the substate lasting tens of seconds to minutes. The effect caused by ryanodol is dependent on transmembrane voltage, with modification more likely to occur and lasting longer at +60 than at -60 mV holding potential. The addition of concentrations of ryanodol insufficient to cause modification does not lead to an increase in single-channel open probability, such as has been reported for ryanodine. At concentrations of > or = 500 mu M, ryanodine after initial rapid modification of the channel leads to irreversible closure

  2. PYRETHROID INSECTICIDES: ISOFORM-DEPENDENT HYDROLYSIS, INDUCTION OF CYTOCHROME P450 3A4 AND EVIDENCE ON THE INVOLVEMENT OF THE PREGNANE X RECEPTOR

    PubMed Central

    Yang, Dongfang; Wang, Xiliang; Chen, Yi-tzai; Deng, Ruitang; Yan, Bingfang

    2009-01-01

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity. PMID:19249324

  3. Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

    SciTech Connect

    Yang Dongfang; Wang Xiliang; Chen Yitzai; Deng Ruitang; Yan Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  4. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    SciTech Connect

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  5. Multiple human isoforms of drug transporters contribute to the hepatic and renal transport of olmesartan, a selective antagonist of the angiotensin II AT1-receptor.

    PubMed

    Yamada, Akihiro; Maeda, Kazuya; Kamiyama, Emi; Sugiyama, Daisuke; Kondo, Tsunenori; Shiroyanagi, Yoshiyuki; Nakazawa, Hayakazu; Okano, Teruo; Adachi, Masashi; Schuetz, John D; Adachi, Yasuhisa; Hu, Zhuohan; Kusuhara, Hiroyuki; Sugiyama, Yuichi

    2007-12-01

    Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with K(m) values of 14.9 and 26.2 microM, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and

  6. Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β1-null Zebrafish Relaxed Is an Exclusive Function of the β1a Subunit*

    PubMed Central

    Schredelseker, Johann; Dayal, Anamika; Schwerte, Thorsten; Franzini-Armstrong, Clara; Grabner, Manfred

    2009-01-01

    The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β1a subunit. Lack of β1a results in (i) reduced membrane expression of the pore forming DHPR α1S subunit, (ii) elimination of α1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β1a from rather general functions of β isoforms. Zebrafish and mammalian β1a subunits quantitatively restored α1S triad targeting and charge movement as well as intracellular Ca2+ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β2a as the phylogenetically closest, and the ancestral housefly βM as the most distant isoform to β1a also completely recovered α1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca2+ transients and very limited tetrad formation compared with β1a. Consequently, larval motility was either only partially restored (β2a-injected larvae) or not restored at all (βM). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β1a isoform. Consequently, we postulate a model that presents β1a as an allosteric modifier of α1S conformation enabling skeletal muscle-type EC coupling. PMID:19008220

  7. Key role of the p110delta isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110delta function in B cells.

    PubMed

    Bilancio, Antonio; Okkenhaug, Klaus; Camps, Montserrat; Emery, Juliet L; Ruckle, Thomas; Rommel, Christian; Vanhaesebroeck, Bart

    2006-01-15

    Mouse gene-targeting studies have documented a central role of the p110delta isoform of phosphoinositide 3-kinase (PI3K) in B-cell development and function. A defect in B-cell antigen receptor (BCR) signaling is key to this B-cell phenotype. Here we further characterize this signaling defect and report that a p110delta-selective small molecule inhibitor mirrors the effect of genetic inactivation of p110delta in BCR signaling. p110delta activity is indispensable for BCR-induced DNA synthesis and phosphorylation of Akt/protein kinase B (PKB), forkhead transcription factor/forkhead box O3a (FOXO3a), and p70 S6 kinase (p70 S6K), with modest effects on the phosphorylation of glycogen synthase kinase 3 alpha/beta (GSK3alpha/beta) and extracellular signal-regulated kinase (Erk). The PI3K-dependent component of intracellular calcium mobilization also completely relies on p110delta catalytic activity. Resting B cells with inactive p110delta fail to enter the cell cycle, correlating with an incapacity to up-regulate the expression of cyclins D2, A, and E, and to phosphorylate the retinoblastoma protein (Rb). p110delta is also critical for interleukin 4 (IL-4)-induced phosphorylation of Akt/PKB and FOXO3a, and protection from apoptosis. Taken together, these data show that defects observed in p110delta mutant mice are not merely a consequence of altered B-cell differentiation, and emphasize the potential utility of p110delta as a drug target in autoimmune diseases in which B cells play a crucial role. PMID:16179367

  8. Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 Isoform*

    PubMed Central

    Zhang, Yuan; Ying, Jiaoqian; Jiang, Dongsheng; Chang, Zhigang; Li, Hua; Zhang, Guoqiang; Gong, Shan; Jiang, Xinghong; Tao, Jin

    2015-01-01

    Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca2+ currents (ICa,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5′-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced ICa,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N′,N′-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCβ1 expression, whereas inhibition of PKCβ1 pharmacologically or by shRNA targeting abolished the U-II-induced ICa,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCβ inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates ICa,L through the βγ subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKCβ1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes. PMID:25678708

  9. Protein 4.1N does not interact with the inositol 1,4,5-trisphosphate receptor in an epithelial cell line.

    PubMed

    Sehgal, Sona; Guerra, Mateus T; Kruglov, Emma A; Wang, Jun; Nathanson, Michael H

    2005-11-01

    Cytosolic Ca2+ regulates a variety of cell functions, and the spatial patterns of Ca2+ signals are responsible in part for the versatility of this second messenger. The subcellular distribution of the inositol 1,4,5-trisphosphate receptor (IP3R) is thought to regulate Ca2+-signaling patterns but little is known about how the distribution of the IP3R itself is regulated. Here we examined the relationship between the IP3R and the cytoskeletal linker protein 4.1N in the polarized WIF-B cell line because protein 4.1N regulates targeting of the type I IP3R in neurons, but WIF-B cells do not express this cytoskeletal protein. WIF-B cells expressed all three isoforms of the IP3R, and each isoform was distributed throughout the cell. These cells did not express the ryanodine receptor. Photorelease of microinjected, caged IP3 induced a rapid rise in cytosolic Ca2+, but the increase began uniformly throughout the cell rather than at a specific initiation site. Expression of protein 4.1N was not associated with redistribution of the IP3R or changes in Ca2+-signaling patterns. These findings are consistent with the hypothesis that the subcellular distribution of IP3R isoforms regulates the formation of Ca2+ waves, and the finding that interactions between protein 4.1N and the IP3R vary among cell types may provide an additional, tissue-specific mechanism to shape the pattern of Ca2+ waves. PMID:16122796

  10. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    SciTech Connect

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  11. Laminin isoforms in endothelial and perivascular basement membranes

    PubMed Central

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  12. Differential regulation of renal phospholipase C isoforms by catecholamines.

    PubMed

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  13. Streaming potentials reveal a short ryanodine-sensitive selectivity filter in cardiac Ca2+ release channel.

    PubMed Central

    Tu, Q; Vélez, P; Brodwick, M; Fill, M

    1994-01-01

    Single cardiac sarcoplasmic reticulum Ca2+ release channels were reconstituted into planar bilayer membranes. Streaming potentials were measured in osmotically asymmetric solutions as a shift in the reversal potential. Potential changes induced by water movement through the bilayer (concentration polarization) and reduced ion activity in the concentrated non-electrolyte solutions were determined using valinomycin. In 400 mM symmetrical CsCH3SO3, the average streaming potential was 2.74 +/- 0.2 mV (n = 5, mean +/- SE; 2 osmol/kg) and independent of the osmoticant used (sucrose or diglycine). Identical streaming potential magnitudes were obtained regardless of which side of the membrane the nonelectrolyte was placed. This suggests that the narrow part of the pore where single file diffusion occurs is relatively short (i.e., accommodates a minimum of 3 H2O molecules). This value is comparable to similar measurements in a variety of surface membrane channels. Ryanodine-modified channels had no measurable streaming potential, an increased Tris+ permeability relative to Cs+, and decreased divalent selectivity (PCs/PTris 5.1 +/- 1.1 to 1.7 +/- 0.3, n = 3; PBa/PCs 8.2 +/- 0.7 to 1.8 +/- 0.5, n = 4). Cation/anion selectivity was essentially unaltered in ryanodine-modified channels. These results suggests that the narrow region of the permeation pathway (i.e., the selectivity filter) is relatively short and widens after ryanodine modification. PMID:7696468

  14. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    PubMed

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  15. Basal activity of GIRK5 isoforms.

    PubMed

    Salvador, Carolina; Mora, Silvia I; Ordaz, Benito; Antaramian, Anaid; Vaca, Luis; Escobar, Laura I

    2003-02-14

    G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results. PMID:12535718

  16. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  17. Cell, Isoform, and Environment Factors Shape Gradients and Modulate Chemotaxis

    PubMed Central

    Chang, S. Laura; Cavnar, Stephen P.; Takayama, Shuichi; Luker, Gary D.; Linderman, Jennifer J.

    2015-01-01

    Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment. PMID:25909600

  18. Alternative splicing results in RET isoforms with distinct trafficking properties

    PubMed Central

    Richardson, Douglas S.; Rodrigues, David M.; Hyndman, Brandy D.; Crupi, Mathieu J. F.; Nicolescu, Adrian C.; Mulligan, Lois M.

    2012-01-01

    RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling. PMID:22875993

  19. Cardiomyocyte ATP Production, Metabolic Flexibility, and Survival Require Calcium Flux through Cardiac Ryanodine Receptors in Vivo*

    PubMed Central

    Bround, Michael J.; Wambolt, Rich; Luciani, Dan S.; Kulpa, Jerzy E.; Rodrigues, Brian; Brownsey, Roger W.; Allard, Michael F.; Johnson, James D.

    2013-01-01

    Ca2+ fluxes between adjacent organelles are thought to control many cellular processes, including metabolism and cell survival. In vitro evidence has been presented that constitutive Ca2+ flux from intracellular stores into mitochondria is required for basal cellular metabolism, but these observations have not been made in vivo. We report that controlled in vivo depletion of cardiac RYR2, using a conditional gene knock-out strategy (cRyr2KO mice), is sufficient to reduce mitochondrial Ca2+ and oxidative metabolism, and to establish a pseudohypoxic state with increased autophagy. Dramatic metabolic reprogramming was evident at the transcriptional level via Sirt1/Foxo1/Pgc1α, Atf3, and Klf15 gene networks. Ryr2 loss also induced a non-apoptotic form of programmed cell death associated with increased calpain-10 but not caspase-3 activation or endoplasmic reticulum stress. Remarkably, cRyr2KO mice rapidly exhibited many of the structural, metabolic, and molecular characteristics of heart failure at a time when RYR2 protein was reduced 50%, a similar degree to that which has been reported in heart failure. RYR2-mediated Ca2+ fluxes are therefore proximal controllers of mitochondrial Ca2+, ATP levels, and a cascade of transcription factors controlling metabolism and survival. PMID:23678000

  20. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. Conclusions These findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans. PMID:24996838

  1. Symmetry-Driven Strategy for the Assembly of the Core Tetracycle of (+)-Ryanodine: Synthetic Utility of a Cobalt-Catalyzed Olefin Oxidation and α-Alkoxy Bridgehead Radical Reaction.

    PubMed

    Nagatomo, Masanori; Hagiwara, Koji; Masuda, Kengo; Koshimizu, Masaki; Kawamata, Takahiro; Matsui, Yuki; Urabe, Daisuke; Inoue, Masayuki

    2016-01-01

    Ryanodine (1) is a potent modulator of intracellular calcium release channels, designated as ryanodine receptors. The exceptionally complex molecular architecture of 1 comprises a highly oxygenated pentacyclic system with eleven contiguous stereogenic centers, which makes it a formidable target for organic synthesis. We identified the embedded C2 -symmetric tricyclic substructure within 1. This specific recognition permitted us to design a concise synthetic route to enantiopure tricycle 9 by utilizing a series of pairwise functionalizations. The four tetrasubstituted carbon centers of 9 were effectively constructed by three key reactions, a dearomatizing Diels-Alder reaction, the kinetic resolution of the obtained racemic 14 through asymmetric methanolysis, and the transannular aldol reaction of the eight-membered diketone 10. A new combination of cobalt-catalyzed hydroperoxidation and NfF-promoted elimination enabled conversion of the hindered olefin of 9 into the corresponding ketone, thus realizing the desymmetrization. Finally, the tetrasubstituted carbon was stereospecifically installed by utilizing the α-alkoxy bridgehead radical to deliver the core tetracycle 7 with the six contiguous tetrasubstituted carbon centers. Consequently, the present work not only accomplishes efficient assembly of four out of the five fused rings of 1, but also develops two new powerful methodologies: two-step ketone formation and bridgehead radical reaction. PMID:26616151

  2. BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

    PubMed

    Foltran, Rocío Beatriz; Diaz, Silvina Laura

    2016-07-01

    The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors. PMID:27167299

  3. Direct Activation of Epac by Sulfonylurea is Isoform Selective

    PubMed Central

    Herbst, Katie J.; Coltharp, Carla; Amzel, L. Mario; Zhang, Jin

    2011-01-01

    Summary Commonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators. PMID:21338921

  4. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  5. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    PubMed

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  6. Multiple effects of ryanodine on intracellular free Ca2+ in smooth muscle cells from bovine and porcine coronary artery: modulation of sarcoplasmic reticulum function.

    PubMed Central

    Wagner-Mann, C.; Hu, Q.; Sturek, M.

    1992-01-01

    1. The effects of ryanodine and caffeine on intracellular free Ca2+ concentration ([Ca2+]i) were studied by use of fura-2 microfluorometry in single smooth muscle cells freshly dispersed from bovine and porcine coronary artery. 2. Bovine and porcine cells demonstrated similar sensitivities to 10 min of exposure to ryanodine in physiological salt solution (PSS), as determined by comparable dose-dependent decreases in the subsequent [Ca2+]i transient induced by 5 mM caffeine. 3. Ryanodine (10 microM) caused a significant increase in [Ca2+]i to a plateau level 27 +/- 3% and 38 +/- 4% above baseline [Ca2+]i (baseline [Ca2+]i = [Ca2+]i at 0 min) in porcine and bovine cells, respectively, when bathed in PSS. In bovine cells the time required to reach 1/2 the plateau level was only 3 min versus 6 min for porcine cells. 4. The ryanodine-induced plateau increase in [Ca2+]i was 35 +/- 5% above baseline for bovine cells bathed in 0 Ca PSS (PSS including 10 microM EGTA with no added Ca2+), but only 7 +/- 3% above baseline in porcine cells during 10 min exposure to 10 microM ryanodine. In bovine cells [Ca2+]i showed proportional increases when extracellular Ca2+ was increased from the normal 2 mM Ca2+ PSS to 5 and 10 mM. 5. Cells pretreated with caffeine in 0 Ca PSS, which depleted the caffeine-sensitive sarcoplasmic reticulum Ca2+ store, showed no increase in [Ca2+]i when challenged with 10 microM ryanodine. The ryanodine-associated increase in [Ca2+]i, which was sustained in 0 Ca PSS during the 10 min ryanodine exposure in cells not pretreated with caffeine, suggests that ryanodine releases Ca2+ from the sarcoplasmic reticulum, but also inhibits Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 PMID:1504718

  7. Estrogen Receptor β Isoform-Specific Induction of Transforming Growth Factor β-Inducible Early Gene-1 in Human Osteoblast Cells: An Essential Role for the Activation Function 1 Domain

    PubMed Central

    Hawse, John R.; Subramaniam, Malayannan; Monroe, David G.; Hemmingsen, Amanda H.; Ingle, James N.; Khosla, Sundeep; Oursler, Merry Jo; Spelsberg, Thomas C.

    2008-01-01

    The estrogen receptors (ER) α and β are important ligand-mediated transcription factors known to play significant biological roles in numerous tissues including bone. Despite the high homology shared by these receptors, recent studies have suggested that their function is largely unique. Although these receptors have been studied in detail for more than a decade, little data exist concerning the mechanisms by which these two proteins regulate distinct sets of genes. Using the TGFβ-inducible early gene-1 (TIEG) as a model, we demonstrate that TIEG is rapidly induced in response to estrogen in osteoblasts by ERβ, but not ERα. We have identified the regulatory elements utilized by ERβ and have demonstrated that ERβ recruits steroid receptor coactivator (SRC)1 and SRC2 to this regulatory region. Additionally, deletion of the ERβ-activation function 1 (AF1) domain drastically decreases the estrogen induction of TIEG. Through the use of chimeric receptors, we have demonstrated that the AF1 domain of ERβ is responsible for recruiting SRC1 and SRC2 and inducing the expression of TIEG in osteoblasts. Finally, SRC1, but not SRC2, is essential for TIEG induction by ERβ. Overall, these data demonstrate that the estrogen induction of TIEG is ERβ specific and that the AF1 domain of ERβ confers this specificity. Finally, a novel and important role for ERβ’s AF1 is implicated in the recruitment of specific coactivators, suggesting that the AF1 may play a significant role in conferring the differences in regulation of gene expression by these two receptors. PMID:18483178

  8. PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation.

    PubMed

    Hands, Katherine J; Cuchet-Lourenco, Delphine; Everett, Roger D; Hay, Ronald T

    2014-01-15

    Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment. PMID:24190887

  9. SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box

    SciTech Connect

    Short, Stephen; Malartre, Marianne; Sharpe, Colin . E-mail: colin.sharpe@port.ac.uk

    2005-09-02

    SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT.

  10. Versican V1 Isoform Induces Neuronal Differentiation and Promotes Neurite Outgrowth

    PubMed Central

    Wu, Yaojiong; Sheng, Wang; Chen, Liwen; Dong, Haiheng; Lee, Vivian; Lu, Fred; Wong, C. Shun; Lu, Wei-Yang; Yang, Burton B.

    2004-01-01

    The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adult brain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth. Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced an aborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failed to do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustained extracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, β1 integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform also promoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versican regulates neuronal development, function, and repair. PMID:14978219

  11. Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during β-adrenergic stimulation

    PubMed Central

    Ferrero, Paola; Said, Matilde; Sánchez, Gina; Vittone, Leticia; Valverde, Carlos; Donoso, Paulina; Mattiazzi, Alicia; Mundiña-Weilenmann, Cecilia

    2007-01-01

    We aimed to define the relative contribution of both PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during β-adrenergic receptor (βAR) stimulation. Rat hearts were perfused with increasing concentrations of the β-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca2+ influx to the cell by low [Ca]o plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [3H]-ryanodine binding and fast Ca2+ release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca2+. Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under βAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the β-induced increase in the channel activity as indicated by the enhancement of the [3H]-ryanodine binding and the velocity of fast SR Ca2+ release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca2+ release during βAR stimulation. PMID:17643448

  12. Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release from the endoplasmic reticulum.

    PubMed

    Guerra-Álvarez, María; Moreno-Ortega, Ana J; Navarro, Elisa; Fernández-Morales, José Carlos; Egea, Javier; López, Manuela G; Cano-Abad, María F

    2015-05-01

    Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2

  13. A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

    PubMed

    Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

    2011-08-01

    The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. PMID:21664424

  14. Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice.

    PubMed

    Moseley, Amy E; Williams, Michael T; Schaefer, Tori L; Bohanan, Cynthia S; Neumann, Jon C; Behbehani, Michael M; Vorhees, Charles V; Lingrel, Jerry B

    2007-01-17

    Several disorders have been associated with mutations in Na,K-ATPase alpha isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimer's disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the alpha1 isoform is found in many cell types, the alpha2 isoform is predominantly expressed in astrocytes, and the alpha3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the alpha2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the alpha3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous alpha1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the alpha2 and alpha3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders. PMID:17234593

  15. Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice.

    PubMed

    Kim, Myung-Hee; de Beer, Maria C; Wroblewski, Joanne M; Charnigo, Richard J; Ji, Ailing; Webb, Nancy R; de Beer, Frederick C; van der Westhuyzen, Deneys R

    2016-06-01

    The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism. PMID:27018443

  16. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  17. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  18. Characterization of a saporin isoform with lower ribosome-inhibiting activity.

    PubMed Central

    Fabbrini, M S; Rappocciolo, E; Carpani, D; Solinas, M; Valsasina, B; Breme, U; Cavallaro, U; Nykjaer, A; Rovida, E; Legname, G; Soria, M R

    1997-01-01

    We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands. PMID:9148741

  19. Quantitative isoform-profiling of highly diversified recognition molecules

    PubMed Central

    Schreiner, Dietmar; Simicevic, Jovan; Ahrné, Erik; Schmidt, Alexander; Scheiffele, Peter

    2015-01-01

    Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands. DOI: http://dx.doi.org/10.7554/eLife.07794.001 PMID:25985086

  20. Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins*

    PubMed Central

    Mehus, Aaron A.; Muhonen, Wallace W.; Garrett, Scott H.; Somji, Seema; Sens, Donald A.; Shabb, John B.

    2014-01-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These

  1. hMENA(11a), a hMENA isoform sending survival signals.

    PubMed

    Trono, Paola; Di Modugno, Francesca; Nisticò, Paola

    2016-03-01

    Human MENA(11a) (hMENA(11a)), an epithelial-associated isoform of the actin binding protein enabled homolog (ENAH, also known as mammalian ENA [MENA]), is upregulated and phosphorylated following the activation of human epidermal growth factor receptor (HER) 1, HER2, and HER3. Here, we reveal a novel role of this isoform in sustaining cell survival and propose hMENA(11a) as a marker of HER3 activation and resistance to phosphatidylinositol-3-kinase inhibition therapies. PMID:27308605

  2. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    PubMed

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions. PMID:26304132

  3. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  4. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  5. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages.

    PubMed

    Papakonstanti, Evangelia A; Zwaenepoel, Olivier; Bilancio, Antonio; Burns, Emily; Nock, Gemma E; Houseman, Benjamin; Shokat, Kevan; Ridley, Anne J; Vanhaesebroeck, Bart

    2008-12-15

    The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer. PMID:19033389

  6. Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

    PubMed Central

    Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

    2015-01-01

    The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

  7. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    PubMed Central

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M.

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  8. Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): Association with aryl hydrocarbon receptor 1 and 2 isoforms

    SciTech Connect

    Lee, Jin-Seon; Kim, Eun-Young Iwata, Hisato

    2009-01-01

    The present study focuses on the molecular mechanism and interspecies differences in susceptibility of avian aryl hydrocarbon receptor (AHR)-cytochrome P4501A (CYP1A) signaling pathway. By the cloning of 5'-flanking regions of CYP1A5 gene from common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus), seven putative xenobiotic response elements (XREs) were identified within 2.7 kb upstream region of common cormorant CYP1A5 (ccCYP1A5), and six XREs were found within 0.9 kb of chicken CYP1A5 (ckCYP1A5). Analysis of sequential deletion and mutagenesis of the binding sites in avian CYP1A5 genes by in vitro reporter gene assays revealed that two XREs at -613 bp and -1585 bp in ccCYP1A5, and one XRE at -262 bp in ckCYP1A5 conferred TCDD-responsiveness. The binding of AHR1 with AHR nuclear translocator 1 (ARNT1) to the functional XRE in a TCDD-dependent manner was verified with gel shift assays, suggesting that avian CYP1A5 is induced by TCDD through AHR1/ARNT1 signaling pathway as well as mammalian CYP1A1 but through a distinct pathway from mammalian CYP1A2, an ortholog of the CYP1A5. TCDD-EC{sub 50} for the transcriptional activity in both cormorant AHR1- and AHR2-ccCYP1A5 reporter construct was 10-fold higher than that in chicken AHR1-ckCYP1A5 reporter construct. In contrast, chicken AHR2 showed no TCDD-dependent response. The TCDD-EC{sub 50} for CYP1A5 transactivation was altered by switching AHR1 between the two avian species, irrespective of the species from which the regulatory region of CYP1A5 gene originates. Therefore, the structural difference in AHR, not the CYP1A5 regulatory region may be a major factor to account for the dioxin susceptibility in avian species.

  9. Differential Roles of Postsynaptic Density-93 Isoforms in Regulating Synaptic Transmission

    PubMed Central

    Krüger, Juliane M.; Favaro, Plinio D.; Liu, Mingna; Kitlińska, Agata; Huang, Xiaojie; Raabe, Monika; Akad, Derya S.; Liu, Yanling; Urlaub, Henning; Dong, Yan; Xu, Weifeng

    2013-01-01

    In the postsynaptic density of glutamatergic synapses, the discs large (DLG)-membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins coordinates a multiplicity of signaling pathways to maintain and regulate synaptic transmission. Postsynaptic density-93 (PSD-93) is the most variable paralog in this family; it exists in six different N-terminal isoforms. Probably because of the structural and functional variability of these isoforms, the synaptic role of PSD-93 remains controversial. To accurately characterize the synaptic role of PSD-93, we quantified the expression of all six isoforms in the mouse hippocampus and examined them individually in hippocampal synapses. Using molecular manipulations, including overexpression, gene knockdown, PSD-93 knock-out mice combined with biochemical assays, and slice electrophysiology both in rat and mice, we demonstrate that PSD-93 is required at different developmental synaptic states to maintain the strength of excitatory synaptic transmission. This strength is differentially regulated by the six isoforms of PSD-93, including regulations of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-active and inactive synapses, and activity-dependent modulations. Collectively, these results demonstrate that alternative combinations of N-terminal PSD-93 isoforms and DLG-MAGUK paralogs can fine-tune signaling scaffolds to adjust synaptic needs to regulate synaptic transmission. PMID:24068818

  10. Osteopontin (OPN/SPP1) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

    PubMed Central

    Lin, Jules; Myers, Amy L.; Wang, Zhuwen; Nancarrow, Derek J.; Ferrer-Torres, Daysha; Handlogten, Amy; Leverenz, Kimmy; Bao, Julia; Thomas, Dafydd G.; Wang, Thomas D.; Orringer, Mark B.; Reddy, Rishindra M.; Chang, Andrew C.; Beer, David G.; Lin, Lin

    2015-01-01

    Esophageal adenocarcinoma (EAC) is often diagnosed at an advanced stage, thus understanding the molecular basis for EAC invasion and metastasis is critical. Here we report that SPP1/OPN was highly overexpressed in primary EACs and intracellularly localized to tumor cells. We further demonstrate that all known OPN isoforms (OPNa, b, c, 4 and 5) were frequently co-overexpressed in primary EACs. Distinct pro-invasion and dissemination phenotypes of isoform-specific OPNb and OPNc stable transfectants were observed. Expression of OPNb significantly enhanced cell migration and adhesion to laminin. In contrast, OPNc cells showed significantly decreased cell migration yet increased cell detachment. Enhanced invasion, both in vitro and in vivo, was observed for OPNb- but not OPNc-expressing cells. Inhibition of RGD integrins, one family of OPN receptors, attenuated OPNb cell migration, abrogated OPNb cell adhesion and significantly reduced OPNb cell clonogenic survival but did not affect OPNc phenotypes, indicating that OPNb but not OPNc acts through integrin-dependent signaling. Differential expression of vimentin, E-cadherin and β-catenin in OPN stable cells may account for the variation in cell adhesion and detachment between these isoforms. We conclude that while all OPN isoforms are frequently co-overexpressed in primary EACs, isoforms OPNb and OPNc enhance invasion and dissemination through collective yet distinct mechanisms. PMID:26068949

  11. Different motifs regulate trafficking of SorCS1 isoforms.

    PubMed

    Nielsen, Morten S; Keat, Sady J; Hamati, Jida W; Madsen, Peder; Gutzmann, Jakob J; Engelsberg, Arne; Pedersen, Karen M; Gustafsen, Camilla; Nykjaer, Anders; Gliemann, Jørgen; Hermans-Borgmeyer, Irm; Kuhl, Dietmar; Petersen, Claus M; Hermey, Guido

    2008-06-01

    The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the alphaC/sigma2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi-endosomal transport to a significant degree. PMID:18315530

  12. ATP- and adenosine-mediated signaling in the central nervous system: adenosine stimulates glutamate release from astrocytes via A2a adenosine receptors.

    PubMed

    Nishizaki, Tomoyuki

    2004-02-01

    Adenosine enhanced intracellular Ca(2+) concentrations in astrocytes via A(2a) adenosine receptors involving protein kinase A (PKA) activation. The Ca(2+) rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP(3)-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A(2a) adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A(2a) adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca(2+) release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes. PMID:14978344

  13. First Trimester Pregnancy Loss and the Expression of Alternatively Spliced NKp30 Isoforms in Maternal Blood and Placental Tissue

    PubMed Central

    Shemesh, Avishai; Tirosh, Dan; Sheiner, Eyal; Benshalom-Tirosh, Neta; Brusilovsky, Michael; Segev, Rotem; Rosental, Benyamin; Porgador, Angel

    2015-01-01

    Capsule: We observed that first trimester pregnancy loss is associated with an altered expression profile of the three isoforms of the NK receptor NKp30 expressed by NKs in PBMC and placental tissue. In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms) in maternal peripheral blood or placental tissue. We conducted a prospective case–control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group comprises women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expressions were mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms-a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. By contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10, and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss. PMID:26082773

  14. Isoform-specific inhibition of ROR alpha-mediated transcriptional activation by human FOXP3.

    PubMed

    Du, Jianguang; Huang, Chunjian; Zhou, Baohua; Ziegler, Steven F

    2008-04-01

    FOXP3 is a forkhead family transcriptional repressor important for the development and function of CD4(+)CD25(+) regulatory T cells. In humans, FOXP3 is expressed as two isoforms, a full-length form and a smaller form lacking exon 2. These two isoforms are expressed in approximately equal amounts in circulating regulatory T cells, and are induced equally in freshly activated CD4(+)CD25(-) T cells. Herein, we show that FOXP3 interacts with retinoic acid receptor-related orphan receptor (ROR)alpha, and that this interaction inhibits transcriptional activation mediated by RORalpha. Full-length FOXP3, but not the isoform lacking exon 2, interacts with RORalpha, and the region of FOXP3 involved in the interaction is encoded by exon 2. Mutation of the LxxLL motif in FOXP3, located in exon 2, abolished interaction and repression by FOXP3. Additionally, the inhibition of RORalpha by FOXP3 does not require an intact forkhead domain, demonstrating a mode of FOXP3 function that is independent of DNA binding. Interestingly, expression of RORalpha in T cells leads to the expression of genes that define Th17 cells, and the expression of each of these gene was inhibited by coexpression of full-length, but not DeltaEx2, FOXP3. These data expand the possible targets of FOXP3-mediated repression and demonstrate functional differences between FOXP3 isoforms. PMID:18354202

  15. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    PubMed Central

    Volpi, Nila; Pecorelli, Alessandra; Lorenzoni, Paola; Di Lazzaro, Francesco; Belmonte, Giuseppe; Aglianò, Margherita; Giannini, Fabio; Grasso, Giovanni

    2013-01-01

    Objective. To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides. PMID:23840094

  16. 45Ca-efflux in embryonic chick heart and its modification by caffeine and ryanodine.

    PubMed

    Prakash, P; Meera, P; Tripathi, O

    1992-12-01

    Ontogenic changes in the kinetics of exchangeable cellular calcium were studied in embryonic (ECV) and post-hatch (PHCV) chick ventricular tissue by monitoring 45Ca-efflux. The isolated whole ventricle (5 & 7 days ECV) or ventricular strips (12 & 18 days ECV and 1-2 days PHCV) were "loaded" with 45Ca (37 degrees C) and then passed through a series of tubes containing efflux solution (4 degrees C) to determine 45Ca-efflux. Curve 'peeling' of the efflux curve indicated existence of 3 kinetically distinct components of exchangeable cellular Ca2+ compartments: C1, C2 & C3. The size of C1, which was the largest in 5 & 7 days ECV decreased significantly to become minimum in 18 days ECV & PHCV. The rate constant of this compartment, however, reduced with the age of the embryo. In contrast, the size of C3 increased with the embryonic development to become the largest in 18 days ECV & PHCV. An increase in the rate constant of this compartment was also observed during embryogenesis. The size and rate constant of C2 remained unaltered during development. However, the increase in size of C3 during embryonic development indicates differentiation of Ca2+ storage sites, like sarcoplasmic reticulum (SR), during the later stages. Caffeine (10 mM) and ryanodine (10 microM) enhanced fractional escape rate during slow phase (ie 120-180 min) of efflux at all developmental stages. The magnitude of enhancement increased during later stages of development indicating greater prominence of SR with the age of embryo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1307380

  17. Smad phospho-isoforms direct context-dependent TGF-β signaling.

    PubMed

    Matsuzaki, Koichi

    2013-08-01

    Better understanding of TGF-β signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-β receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-β change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-β signaling. PMID:23871609

  18. Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone

    PubMed Central

    Li, Kang; Guo, Enen; Hossain, Muktadir S.; Li, Qingrong; Cao, Yang; Tian, Ling; Deng, Xiaojuan; Li, Sheng

    2015-01-01

    Resulted from alternative splicing of the 5′ exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5th larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5th larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level. PMID:26166384

  19. Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

    PubMed

    Li, Kang; Guo, Enen; Hossain, Muktadir S; Li, Qingrong; Cao, Yang; Tian, Ling; Deng, Xiaojuan; Li, Sheng

    2015-01-01

    Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level. PMID:26166384

  20. Effectiveness of GH isoform differential immunoassay for detecting rhGH doping on application of various growth factors.

    PubMed

    Okano, Masato; Nishitani, Yasunori; Sato, Mitsuhiko; Kageyama, Shinji

    2012-09-01

    The analytical method for detecting growth hormone (GH) doping, the so-called GH isoform differential immunoassay, is currently approved by the World Anti-Doping Agency (WADA). Anti-doping laboratories often face challenges by athletes' lawyers and need to have various types of scientific evidence against the claim that the adverse analytical finding (AAF) result was caused by excess ectopic or abnormal excretion. In this work, a population study of Japanese athletes (255 male and 256 female) and administration studies of recombinant human GH (rhGH) in Japanese females were conducted to confirm the applicability of GH isoform differential immunoassay. The present paper describes the effectiveness of the GH isoform differential immunoassay under abnormal excretion of endogenous GH as determined by administration studies of GH releasing hormone (GHRH(1-44)) and insulin-like growth factor-1 (IGF-1). No false positive findings were found in Japanese athletes. The GH isoform differential immunoassays could detect application of rhGH for approximately 12-24 h. The administration of GHRH(1-44) and IGF-1 as well as ghrelin receptor agonists did not affect the isoform ratio (no false positives). We conclude that the GH isoform differential immunoassay is a highly specific method for detecting rhGH doping. Subject-based profiling (i.e. athlete biological passport) very likely will represent a highly sensitive approach for detecting rhGH doping. PMID:22733714

  1. Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system.

    PubMed

    Dickinson, Patsy S; Kurland, Sienna C; Qu, Xuan; Parker, Brett O; Sreekrishnan, Anirudh; Kwiatkowski, Molly A; Williams, Alex H; Ysasi, Alexandra B; Christie, Andrew E

    2015-09-01

    Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family. PMID:26206359

  2. Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system

    PubMed Central

    Dickinson, Patsy S.; Kurland, Sienna C.; Qu, Xuan; Parker, Brett O.; Sreekrishnan, Anirudh; Kwiatkowski, Molly A.; Williams, Alex H.; Ysasi, Alexandra B.; Christie, Andrew E.

    2015-01-01

    ABSTRACT Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family. PMID:26206359

  3. Differential expression of vascular endothelial growth factor-A isoforms in the mouse uterus during early pregnancy.

    PubMed

    Walter, Lisa M; Rogers, Peter A W; Girling, Jane E

    2010-12-01

    While vascular endothelial growth factor (VEGF)-A mediates endometrial vascular remodelling during early pregnancy in mice, individual VEGF-A isoforms have not been investigated, despite their different biological properties. Using mice as a model, the expression of VEGF-A isoforms and receptors in the mouse uterus during early pregnancy was quantified. It was postulated that selected isoform expression would increase concurrent with increased endometrial endothelial cell proliferation at this time. Uteri were collected on days 1-5 of pregnancy and mRNA expression was quantified by quantitative reverse-transcription polymerase chain reaction, VEGF-A protein by Western blot and VEGF receptor (VEGFR)-2 by immunohistochemistry. The lowest expression of isomers Vegf(120) and Vegf(164) was observed on day 2 of pregnancy, increasing thereafter. Vegfr-2 mRNA expression was significantly higher on days 3-5 of pregnancy relative to days 1-2 (P<0.05). No significant changes were noted in Vegf(188), Nrp1 or Nrp2 mRNA. VEGF(188) protein expression was consistently higher than other isoforms. These data demonstrate differential regulation of VEGF-A isoforms in mouse uterus during early pregnancy. PMID:21050818

  4. Frataxin mRNA isoforms in FRDA patients and normal subjects: effect of tocotrienol supplementation.

    PubMed

    Abruzzo, Provvidenza Maria; Marini, Marina; Bolotta, Alessandra; Malisardi, Gemma; Manfredini, Stefano; Ghezzo, Alessandro; Pini, Antonella; Tasco, Gianluca; Casadio, Rita

    2013-01-01

    Friedreich's ataxia (FRDA) is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3) in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor-γ (PPARG), PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role. PMID:24175286

  5. Both Myosin-10 isoforms are required for radial neuronal migration in the developing cerebral cortex.

    PubMed

    Ju, Xing-Da; Guo, Ye; Wang, Nan-Nan; Huang, Ying; Lai, Ming-Ming; Zhai, Yan-Hua; Guo, Yu-Guang; Zhang, Jian-Hua; Cao, Rang-Juan; Yu, Hua-Li; Cui, Lei; Li, Yu-Ting; Wang, Xing-Zhi; Ding, Yu-Qiang; Zhu, Xiao-Juan

    2014-05-01

    During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration. PMID:23300110

  6. Developmental exposure to polychlorinated biphenyls (PCBs) interferes with experience-dependent dendritic plasticity and ryanodine receptor expression in weanling rats.

    EPA Science Inventory

    BACKGROUND: Neurodevelopmental disorders are associated with altered patterns of neuronal connectivity. A critical determinant of neuronal connectivity is the dendritic morphology of individual neurons, which is shaped by experience. The identification of environmental exposures ...

  7. Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemia-reperfusion

    PubMed Central

    Fauconnier, Jérémy; Meli, Albano C.; Thireau, Jérôme; Roberge, Stephanie; Shan, Jian; Sassi, Yassine; Reiken, Steven R.; Rauzier, Jean-Michel; Marchand, Alexandre; Chauvier, David; Cassan, Cécile; Crozier, Christine; Bideaux, Patrice; Lompré, Anne-Marie; Jacotot, Etienne; Marks, Andrew R.; Lacampagne, Alain

    2011-01-01

    Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca2+ and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca2+ homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca2+ channel stabilizer S107 (“rycal”) inhibited the SR Ca2+ leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α–induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca2+ leak trough normalization of RyR2 function is cardioprotective. PMID:21788490

  8. The three-dimensional structural surface of two beta-sheet scorpion toxins mimics that of an alpha-helical dihydropyridine receptor segment.

    PubMed Central

    Green, Daniel; Pace, Suzi; Curtis, Suzanne M; Sakowska, Magdalena; Lamb, Graham D; Dulhunty, Angela F; Casarotto, Marco G

    2003-01-01

    An alpha-helical II-III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonist's activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native beta-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity alpha-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the alpha-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2-20-fold increase in the activity of the alpha-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified alpha-helical peptide and the beta-sheet toxin, a 10-100-fold reduction in channel closed time, with a < or = 2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites. PMID:12429019

  9. Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes*

    PubMed Central

    Smith, Gina A.; Howell, Gareth J.; Phillips, Clair; Muench, Stephen P.; Ponnambalam, Sreenivasan; Harrison, Michael A.

    2016-01-01

    Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a1 and a3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a1 and a3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype. PMID:26912656

  10. Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting.

    PubMed

    St John, P L; Wang, R; Yin, Y; Miner, J H; Robert, B; Abrahamson, D R

    2001-04-01

    Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo. PMID:11249861

  11. Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

    PubMed Central

    Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

    2015-01-01

    The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

  12. Redundancy of IL-1 Isoform Signaling and Its Implications for Arterial Remodeling

    PubMed Central

    Beltrami-Moreira, Marina; Vromman, Amélie; Sukhova, Galina K.; Folco, Eduardo J.; Libby, Peter

    2016-01-01

    Aims Mice deficient in IL-1 receptor 1 (hence unresponsive to both IL-1 isoforms α and β) have impaired expansive arterial remodeling due to diminished expression of matrix-degrading enzymes, especially MMP-3. Emergence of IL-1 as a target in cardiovascular disease prompted the investigation of the redundancy of IL-1α and IL-1β in the induction of MMP-3 and other matrix-remodeling enzymes in human cells. Methods and Results Human primary vascular smooth muscle cells (VSMCs) and carotid endarterectomy specimens were stimulated with equimolar concentrations of IL-1α or IL-1β and analyzed protease expression by immunoblot and ELISA. Either IL-1α or IL-1β increased the expression of pro-MMP-3 in VSMCs, facilitated VSMC migration through Matrigel, and induced MMP-3 production in specimens from atheromatous plaques. VSMCs also secreted MMP-1 and Cathepsin S (CatS) upon stimulation with IL-1α or IL-1β. IL-1 isoforms similarly increased MMP-1 and MMP-9 expression in carotid endarterectomy specimens. We examined the expression of MMP-3 and IL-1 isoforms by immunostaining of carotid atheromata, calculated the % positive areas, and tested associations by linear regression. MMP-3 colocalized with IL-1 isoforms in atheromata. MMP-3+ area in plaques positively associated with IL-1α+ (R2 = 0.61, P<0.001) and with IL-1β + areas (R2 = 0.68, P<0.001). MMP-3+ area within atheroma also associated with CD68+ area, but not with α-smooth muscle actin area. Conclusions Either IL-1α or IL-1β can induce the expression of enzymes implicated in remodeling of the arterial extracellular matrix, and facilitate human VSMC migration in vitro. Human atheromata contain both IL-1 isoforms in association with immunoreactive MMP-3. This redundancy of IL-1 isoforms suggests that selective blocking of one IL-1 isoform should not impair expansive arterial remodeling, a finding with important clinical implications for therapeutic targeting of IL-1 in atherosclerosis. PMID:27032103

  13. EXPERIENCE-DEPENDENT REGULATION OF TRKB ISOFORMS IN RODENT VISUAL CORTEX

    PubMed Central

    Bracken, Bethany K.; Turrigiano, Gina G.

    2010-01-01

    Within primary visual cortex (V1), BDNF signaling through its high affinity receptor TrkB is important for normal development and experience-dependent plasticity. TrkB is expressed in several alternatively spliced isoforms, including full length TrkB (TrkB.FL), and several truncated isoforms (TrkB.T1, TrkB.T2 and TrkB.T4) that lack the intracellular tyrosine kinase domain. These isoforms are important components of BDNF signaling, yet little is known about the developmental or experience-dependent regulation of their expression. Using immunohistochemistry, we found TrkB.FL and TrkB.T1 expressed in interneurons and pyramidal neurons within V1, but not in cortical astrocytes. We used real-time PCR to quantify changes in mRNA expression of BDNF, the four TrkB isoforms, and the low affinity receptor P75NTR during normal development, and in response to visual deprivation at two different ages. BDNF expression increased between postnatal days 10 (P10) and P30, and was rapidly down-regulated by 3 days of visual deprivation during both the pre-critical period (P14–P17) and the critical period (P18–P21). Over the same developmental period expression of each TrkB isoform was regulated independently; TrkB.T1 increased, TrkB.FL and TrkB.T2 decreased, and TrkB.T4 showed transient changes. Neither brief visual deprivation nor prolonged dark-rearing induced changes in TrkB.FL or TrkB.T1 expression. However, TrkB.T4 expression was reduced by brief visual deprivation, while TrkB.T4, TrkB.T2 and P75NTR were up-regulated by prolonged dark-rearing into the critical period. Our data indicate that TrkB isoform expression can be selectively regulated by visual experience, and may contribute to experience-dependent cortical plasticity. PMID:19224567

  14. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

    Ivanov, Sergey V.; Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I.

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  15. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  16. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle

    SciTech Connect

    Chadwick, C.C.; Saito, A.; Fleischer, S. )

    1990-03-01

    The release of Ca{sup 2+} from internal stores is requisite to muscle contraction. In skeletal muscle and heart, the Ca{sup 2+} release channels (ryanodine receptor) of sarcoplasmic reticulum, involved in excitation-contraction coupling, have recently been isolated and characterized. In smooth muscle, inositol 1,4,5-trisphosphate (IP{sub 3}) is believed to mobilize Ca{sup 2+} from internal stores and thereby modulate contraction. The authors describe the isolation of an IP{sub 3} receptor from smooth muscle. Bovine aorta smooth muscle microsomes were solubilized with 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, and the IP{sub 3} receptor was purified by sucrose gradient centrifugation and column chromatography with heparin-agarose and wheat germ agglutinin-agarose. The receptor is an oligomer of a single polypeptide with a M{sub r} of 224,000 as determined by SDS/PAGE. Negative-staining electron microscopy reveals that the receptor is a large pinwheel-like structure having surface dimensions of {approx}250 {times} 250 {angstrom} with fourfold symmetry. The IP{sub 3} receptor from smooth muscle is similar to the ryanodine receptor with regard to its large size and fourfold symmetry, albeit distinct with regard to appearance, protomer size, and ligand binding.

  17. Decoding RAS isoform and codon-specific signalling

    PubMed Central

    Newlaczyl, Anna U.; Hood, Fiona E.; Coulson, Judy M.; Prior, Ian A.

    2014-01-01

    RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms. PMID:25109951

  18. Separation of plasmid DNA isoforms using centrifugal ultrafiltration.

    PubMed

    Borujeni, Ehsan Espah; Zydney, Andrew L

    2012-07-01

    Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms. PMID:22780319

  19. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function. PMID:27226544

  20. CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

    PubMed Central

    Krzywinska, Ewelina; Cornillon, Amelie; Allende-Vega, Nerea; Vo, Dang-Nghiem; Rene, Celine; Lu, Zhao-Yang; Pasero, Christine; Olive, Daniel; Fegueux, Nathalie; Ceballos, Patrick; Hicheri, Yosr; Sobecki, Michal; Rossi, Jean-François; Cartron, Guillaume; Villalba, Martin

    2016-01-01

    The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56bright, NK cells. Most NK cells in healthy human donors are CD45RA+CD45RO-. The CD45RA-RO+ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RAdimRO- (CD45RAdim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RAdim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RAdim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells. PMID:27100180

  1. Human Dectin-1 isoform E is a cytoplasmic protein and interacts with RanBPM.

    PubMed

    Xie, Jianhui; Sun, Maoyun; Guo, Liang; Liu, Weicheng; Jiang, Jianhai; Chen, Xiaoning; Zhou, Lei; Gu, Jianxin

    2006-09-01

    Human Dectin-1, a type II transmembrane receptor, is alternatively spliced, generating eight isoforms. Of these isoforms, the isoform E (hDectin-1E) is structurally unique, containing a complete C-type lectin-like domain as well as an ITAM-like sequence. So far, little is known about its function. In the present study, we demonstrated that hDectin-1E was not secreted and it mainly resided in the cytoplasm. Using yeast two-hybrid screening, we identified a Ran-binding protein, RanBPM, as an interacting partner of hDectin-1E. GST pull-down assays showed that RanBPM interacted directly with hDectin-1E and the region containing SPRY domain was sufficient for the interaction. The binding of hDectin-1E and RanBPM was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Taken together, our data provide a clue to the understanding of the function about hDectin-1E. PMID:16870151

  2. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

    PubMed

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. PMID:26170631

  3. Alzheimer's disease is associated with low density of the long CR1 isoform.

    PubMed

    Mahmoudi, Rachid; Kisserli, Aymric; Novella, Jean-Luc; Donvito, Béatrice; Dramé, Moustapha; Réveil, Brigitte; Duret, Valérie; Jolly, Damien; Pham, Bach-Nga; Cohen, Jacques H

    2015-04-01

    The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation. PMID:25666996

  4. Measurement of calcium release due to inositol trisphosphate receptors in skeletal muscle.

    PubMed

    Casas, Mariana; Altamirano, Francisco; Jaimovich, Enrique

    2012-01-01

    Calcium transients elicited by IP(3) receptors upon electrical stimulation of skeletal muscle cells (slow calcium signals) are often hard to visualize due to their relatively small amplitude compared to the large transient originated from ryanodine receptors associated to excitation-contraction coupling. The study of slow calcium transients, however, is relevant due to their function in regulation of muscle gene expression and in the process of excitation-transcription coupling. Discussed here are the procedures used to record slow calcium signals from both cultured mouse myotubes and from cultured adult skeletal muscle fibers. PMID:22130849

  5. Peritumoral Small EphrinA5 Isoform Level Predicts the Postoperative Survival in Hepatocellular Carcinoma

    PubMed Central

    Yeh, Ta-Sen; Wang, Yu-Ling; Liang, Kung-Hao; Yeh, Chau-Ting; Chen, Tse-Ching

    2012-01-01

    Background EphrinA5, a member of Eph/Ephrin family, possesses two alternative isoforms, large ephrinA5 isoform (ephrinA5L) and small ephrinA5 isoform (ephrinA5S). EphrinA5L is a putative tumor suppressor in several types of human cancers. However, the role of ephrinA5S in hepato-carcinogenesis remains unclear. In this study, we evaluate the role of ephrinA5 isoforms in human hepatocellular carcinomas (HCC). Methodology/Principal Findings A total of 142 paired HCCs and peritumoral liver tissue was examined for relative expression of ephrinA5L and ephrinA5S by using quantitative real-time polymerase chain reaction. We analyzed their expression in relation to clinical parameters, disease-free survival and overall survival. Functional assays were performed to dissect the possible underlying mechanisms. Both ephrinA5L and ephrinA5S were significantly downregulated in HCCs, as compared to those in peritumoral tissue (p = 0.013 and 0.001). Univariate analysis demonstrated that ephrinA5S was positively correlated with old age and histological grade. In multivariate analysis, high ephrinA5S expression in peritumoral tissue had better disease-free survival (p = 0.002) and overall survival (p = 0.045) in patients with HCC after surgical resection. Functional analysis in HCC cell lines revealed that ephrinA5S had a more potent suppressive effect than ephrinA5L on cell proliferation (p<0.05) and migration (p<0.01). Furthermore, forced expression of both ephrinA5 isoforms in HCC cell lines significantly down-regulated epidermal growth factor receptor (EGFR) expression by promoting c-Cbl-mediated EGFR degradation. Conclusions/Significance EphrinA5S might be a useful prognostic biomarker for HCCs after surgical resection. EphrinA5, especially ephrinA5S, acts as a tumor suppressor in hepatocarcinogenesis. Peritumoral small ephrinA5 isoform level could determine the postoperative survival in hepatocellular carcinoma. PMID:22860012

  6. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  7. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  8. IL-33 isoforms: their future as vaccine adjuvants?

    PubMed Central

    Villarreal, Daniel O; Weiner, David B

    2015-01-01

    The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy. PMID:25656504

  9. Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

    PubMed

    Azzi, Sandy; Gallerne, Cindy; Romei, Cristina; Le Coz, Vincent; Gangemi, Rosaria; Khawam, Krystel; Devocelle, Aurore; Gu, Yanhong; Bruno, Stefania; Ferrini, Silvano; Chouaib, Salem; Eid, Pierre; Azzarone, Bruno; Giron-Michel, Julien

    2015-06-01

    Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression. PMID:26152359

  10. The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

    SciTech Connect

    Mukhopadhyay, Sudit S. . E-mail: suditmukhopadhy@yahoo.com; Rosen, Jeffrey M. . E-mail: jrosen@bcm.tmc.edu

    2007-07-06

    The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5.

  11. The γ-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane

    PubMed Central

    Mah, Kar Men; Houston, Douglas W.; Weiner, Joshua A.

    2016-01-01

    The 22 γ-Protocadherin (γ-Pcdh) adhesion molecules encoded by the Pcdhg gene cluster play critical roles in nervous system development, including regulation of dendrite arborisation, neuronal survival, and synaptogenesis. Recently, they have been implicated in suppression of tumour cell growth by inhibition of canonical Wnt signalling, though the mechanisms through which this occurs remain unknown. Here, we show differential regulation of Wnt signalling by individual γ-Pcdhs: The C3 isoform uniquely inhibits the pathway, whilst 13 other isoforms upregulate signalling. Focusing on the C3 isoform, we show that its unique variable cytoplasmic domain (VCD) is the critical one for Wnt pathway inhibition. γ-Pcdh-C3, but not other isoforms, physically interacts with Axin1, a key component of the canonical Wnt pathway. The C3 VCD competes with Dishevelled for binding to the DIX domain of Axin1, which stabilizes Axin1 at the membrane and leads to reduced phosphorylation of Wnt co-receptor Lrp6. Finally, we present evidence that Wnt pathway activity can be modulated up (by γ-Pcdh-A1) or down (by γ-Pcdh-C3) in the cerebral cortex in vivo, using conditional transgenic alleles. Together, these data delineate opposing roles for γ-Pcdh isoforms in regulating Wnt signalling and identify Axin1 as a novel protein interactor of the widely-expressed γ-Pcdh-C3 isoform. PMID:27530555

  12. Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

    PubMed

    Gong, Yan; Bourhis, Eric; Chiu, Cecilia; Stawicki, Scott; DeAlmeida, Venita I; Liu, Bob Y; Phamluong, Khanhky; Cao, Tim C; Carano, Richard A D; Ernst, James A; Solloway, Mark; Rubinfeld, Bonnee; Hannoush, Rami N; Wu, Yan; Polakis, Paul; Costa, Mike

    2010-01-01

    β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for

  13. 20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx.

    PubMed

    Li, Kang; Tian, Ling; Guo, Zhongjian; Guo, Sanyou; Zhang, Jianzhen; Gu, Shi-Hong; Palli, Subba R; Cao, Yang; Li, Sheng

    2016-08-26

    The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing. PMID:27365399

  14. Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing

    PubMed Central

    Sun, Wei; You, Xintian; Gogol-Döring, Andreas; He, Haihuai; Kise, Yoshiaki; Sohn, Madlen; Chen, Tao; Klebes, Ansgar; Schmucker, Dietmar; Chen, Wei

    2013-01-01

    The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18 496 isoforms, out of 19 008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons. PMID:23792425

  15. Regulation and Functional Implications of Opioid Receptor Splicing in Opioid Pharmacology and HIV Pathogenesis.

    PubMed

    Regan, Patrick M; Langford, Dianne; Khalili, Kamel

    2016-05-01

    Despite the identification and characterization of four opioid receptor subtypes and the genes from which they are encoded, pharmacological data does not conform to the predications of a four opioid receptor model. Instead, current studies of opioid pharmacology suggest the existence of additional receptor subtypes; however, no additional opioid receptor subtype has been identified to date. It is now understood that this discrepancy is due to the generation of multiple isoforms of opioid receptor subtypes. While several mechanisms are utilized to generate these isoforms, the primary mechanism involves alternative splicing of the pre-mRNA transcript. Extensive alternative splicing patterns for opioid receptors have since been identified and discrepancies in opioid pharmacology are now partially attributed to variable expression of these isoforms. Recent studies have been successful in characterizing the localization of these isoforms as well as their specificity in ligand binding; however, the regulation of opioid receptor splicing specificity is poorly characterized. Furthermore, the functional significance of individual receptor isoforms and the extent to which opioid- and/or HIV-mediated changes in the opioid receptor isoform profile contributes to altered opioid pharmacology or the well-known physiological role of opioids in the exacerbation of HIV neurocognitive dysfunction is unknown. As such, the current review details constitutive splicing mechanisms as well as the specific architecture of opioid receptor