Sample records for gap junctional channels

  1. Hexadecameric structure of an invertebrate gap junction channel.

    PubMed

    Oshima, Atsunori; Matsuzawa, Tomohiro; Murata, Kazuyoshi; Tani, Kazutoshi; Fujiyoshi, Yoshinori

    2016-03-27

    Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Measurement of Single Channel Currents from Cardiac Gap Junctions

    NASA Astrophysics Data System (ADS)

    Veenstra, Richard D.; Dehaan, Robert L.

    1986-08-01

    Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

  3. Intracellular trafficking pathways of Cx43 gap junction channels.

    PubMed

    Epifantseva, Irina; Shaw, Robin M

    2018-01-01

    Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Gap Junctions

    PubMed Central

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2013-01-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

  5. Electrical transmission between mammalian neurons is supported by a small fraction of gap junction channels.

    PubMed

    Curti, Sebastian; Hoge, Gregory; Nagy, James I; Pereda, Alberto E

    2012-06-01

    Electrical synapses formed by gap junctions between neurons create networks of electrically coupled neurons in the mammalian brain, where these networks have been found to play important functional roles. In most cases, interneuronal gap junctions occur at remote dendro-dendritic contacts, making difficult accurate characterization of their physiological properties and correlation of these properties with their anatomical and morphological features of the gap junctions. In the mesencephalic trigeminal (MesV) nucleus where neurons are readily accessible for paired electrophysiological recordings in brain stem slices, our recent data indicate that electrical transmission between MesV neurons is mediated by connexin36 (Cx36)-containing gap junctions located at somato-somatic contacts. We here review evidence indicating that electrical transmission between these neurons is supported by a very small fraction of the gap junction channels present at cell-cell contacts. Acquisition of this evidence was enabled by the unprecedented experimental access of electrical synapses between MesV neurons, which allowed estimation of the average number of open channels mediating electrical coupling in relation to the average number of gap junction channels present at these contacts. Our results indicate that only a small proportion of channels (~0.1 %) appear to be conductive. On the basis of similarities with other preparations, we postulate that this phenomenon might constitute a general property of vertebrate electrical synapses, reflecting essential aspects of gap junction function and maintenance.

  6. A structural and functional comparison of gap junction channels composed of connexins and innexins

    PubMed Central

    Williams, Jamal B.

    2016-01-01

    ABSTRACT Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre‐chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue‐ and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure–function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin‐based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522–547, 2017 PMID:27582044

  7. Defining the factors that affect solute permeation of gap junction channels.

    PubMed

    Valiunas, Virginijus; Cohen, Ira S; Brink, Peter R

    2018-01-01

    This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin

    PubMed Central

    Fontes, Joseph D.; Ramsey, Jon; Polk, Jeremy M; Koop, Andre; Denisova, Janna V.; Belousov, Andrei B.

    2015-01-01

    Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed. PMID:26017008

  9. A structural and functional comparison of gap junction channels composed of connexins and innexins.

    PubMed

    Skerrett, I Martha; Williams, Jamal B

    2017-05-01

    Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre-chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue- and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure-function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin-based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522-547, 2017. © 2016 The Authors Developmental Neurobiology Published by Wiley Periodicals, Inc.

  10. An electrostatic mechanism for Ca2+-mediated regulation of gap junction channels

    PubMed Central

    Bennett, Brad C.; Purdy, Michael D.; Baker, Kent A.; Acharya, Chayan; McIntire, William E.; Stevens, Raymond C.; Zhang, Qinghai; Harris, Andrew L.; Abagyan, Ruben; Yeager, Mark

    2016-01-01

    Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca2+ blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca2+. The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca2+ coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca2+chelation. Computational analysis revealed that Ca2+-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K+ into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore. PMID:26753910

  11. Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

    PubMed Central

    Starich, Todd A.; Hall, David H.; Greenstein, David

    2014-01-01

    In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans. PMID:25195067

  12. Monovalent Cation Permeation through the Connexin40 Gap Junction Channel

    PubMed Central

    Beblo, Dolores A.; Veenstra, Richard D.

    1997-01-01

    The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius. PMID:9101408

  13. Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

    PubMed

    Starich, Todd A; Hall, David H; Greenstein, David

    2014-11-01

    In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma-germline interactions in C. elegans. Copyright © 2014 by the Genetics Society of America.

  14. Adrenocortical Gap Junctions and Their Functions

    PubMed Central

    Bell, Cheryl L.; Murray, Sandra A.

    2016-01-01

    Adrenal cortical steroidogenesis and proliferation are thought to be modulated by gap junction-mediated direct cell–cell communication of regulatory molecules between cells. Such communication is regulated by the number of gap junction channels between contacting cells, the rate at which information flows between these channels, and the rate of channel turnover. Knowledge of the factors regulating gap junction-mediated communication and the turnover process are critical to an understanding of adrenal cortical cell functions, including development, hormonal response to adrenocorticotropin, and neoplastic dedifferentiation. Here, we review what is known about gap junctions in the adrenal gland, with particular attention to their role in adrenocortical cell steroidogenesis and proliferation. Information and insight gained from electrophysiological, molecular biological, and imaging (immunocytochemical, freeze fracture, transmission electron microscopic, and live cell) techniques will be provided. PMID:27445985

  15. The gap junction channel protein connexin 43 is covalently modified and regulated by SUMOylation.

    PubMed

    Kjenseth, Ane; Fykerud, Tone A; Sirnes, Solveig; Bruun, Jarle; Yohannes, Zeremariam; Kolberg, Matthias; Omori, Yasufumi; Rivedal, Edgar; Leithe, Edward

    2012-05-04

    SUMOylation is a posttranslational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in specific target proteins. Most known SUMOylation target proteins are located in the nucleus, but there is increasing evidence that SUMO may also be a key determinant of many extranuclear processes. Gap junctions consist of arrays of intercellular channels that provide direct transfer of ions and small molecules between adjacent cells. Gap junction channels are formed by integral membrane proteins called connexins, of which the best-studied isoform is connexin 43 (Cx43). Here we show that Cx43 is posttranslationally modified by SUMOylation. The data suggest that the SUMO system regulates the Cx43 protein level and the level of functional Cx43 gap junctions at the plasma membrane. Cx43 was found to be modified by SUMO-1, -2, and -3. Evidence is provided that the membrane-proximal lysines at positions 144 and 237, located in the Cx43 intracellular loop and C-terminal tail, respectively, act as SUMO conjugation sites. Mutations of lysine 144 or lysine 237 resulted in reduced Cx43 SUMOylation and reduced Cx43 protein and gap junction levels. Altogether, these data identify Cx43 as a SUMOylation target protein and represent the first evidence that gap junctions are regulated by the SUMO system.

  16. Connexin40 and connexin43 determine gating properties of atrial gap junction channels.

    PubMed

    Lin, Xianming; Gemel, Joanna; Glass, Aaron; Zemlin, Christian W; Beyer, Eric C; Veenstra, Richard D

    2010-01-01

    While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance (g(j)). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g(j). Slightly more than half of atrial g(j) and gap junctions, the transjunctional voltage (V(j))-dependent inactivation of atrial g(j) was reduced and kinetically slowed, while the V(j)-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g(j). Co-expression of Cx40 accounts for most, but not all, of the differences in the V(j)-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias. Copyright 2009 Elsevier Inc. All rights reserved.

  17. Ionic blockade of the rat connexin40 gap junction channel by large tetraalkylammonium ions.

    PubMed

    Musa, H; Gough, J D; Lees, W J; Veenstra, R D

    2001-12-01

    The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.

  18. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification.

    PubMed

    Alqadah, Amel; Hsieh, Yi-Wen; Schumacher, Jennifer A; Wang, Xiaohong; Merrill, Sean A; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.

  19. [Influence of Cx26/Cx32 gap junction channel on antineoplastic effect of etoposide in Hela cells].

    PubMed

    Tong, Xu-Hui; Dong, Shu-Ying; Jiang, Guo-Jun; Fan, Gao-Fu

    2012-03-01

    To observe the influence of Cx26/Cx32 gap junction channel on the antineoplastic effect of etoposide in Hela cervical cancer cells. Fluorescence trace was used to assay the gap junction intercellular communication mediated by Cx26/Cx32 in Hela cells and its functional modulation by the pharmacological agents (oleamide, retinoid acid). A standard colony-forming assay was applied to determine the cell growth-inhibiting effect of etoposide in Hela cells with functional modulation of the gap junction. Hoechst 33258 staining was used to assess the changes in etoposide-induced apoptosis of Hela cells with altered gap junction functions. Oleamide markedly decreased while retinoid acid obviously increased the gap junction function in Hela cells. Standard colony-forming assay showed that etoposide produced a lowered antiproliferative effect in Hela cells with reduced gap junction and an increased antiproliferative effect in cells with enhanced gap junction function. In cells with a reduced gap junction function, etoposide induced a lowered apoptosis rate, which increased obviously in cells with an enhanced gap junction function. The antineoplastic effect of etoposide is reduced in Hela cells with a decreased gap junction intercellular communication mediated by Cx26/Cx32 and is enhanced in cells with an increased gap junction intercellular communication.

  20. Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

    PubMed

    Pogoda, Kristin; Kameritsch, Petra; Retamal, Mauricio A; Vega, José L

    2016-05-24

    Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.

  1. Joint diseases: from connexins to gap junctions.

    PubMed

    Donahue, Henry J; Qu, Roy W; Genetos, Damian C

    2017-12-19

    Connexons form the basis of hemichannels and gap junctions. They are composed of six tetraspan proteins called connexins. Connexons can function as individual hemichannels, releasing cytosolic factors (such as ATP) into the pericellular environment. Alternatively, two hemichannel connexons from neighbouring cells can come together to form gap junctions, membrane-spanning channels that facilitate cell-cell communication by enabling signalling molecules of approximately 1 kDa to pass from one cell to an adjacent cell. Connexins are expressed in joint tissues including bone, cartilage, skeletal muscle and the synovium. Indicative of their importance as gap junction components, connexins are also known as gap junction proteins, but individual connexin proteins are gaining recognition for their channel-independent roles, which include scaffolding and signalling functions. Considerable evidence indicates that connexons contribute to the function of bone and muscle, but less is known about the function of connexons in other joint tissues. However, the implication that connexins and gap junctional channels might be involved in joint disease, including age-related bone loss, osteoarthritis and rheumatoid arthritis, emphasizes the need for further research into these areas and highlights the therapeutic potential of connexins.

  2. Quantification of gap junction selectivity.

    PubMed

    Ek-Vitorín, Jose F; Burt, Janis M

    2005-12-01

    Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance (gj) for each junction studied, such that the selective permeability (k2-DYE/gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower.

  3. Relative Roles of Gap Junction Channels and Cytoplasm in Cell-to-Cell Diffusion of Fluorescent Tracers

    NASA Astrophysics Data System (ADS)

    Safranyos, Richard G. A.; Caveney, Stanley; Miller, James G.; Petersen, Nils O.

    1987-04-01

    Intercellular (tissue) diffusion of molecules requires cytoplasmic diffusion and diffusion through gap junctional (or cell-to-cell) channels. The rates of tissue and cytoplasmic diffusion of fluorescent tracers, expressed as an effective diffusion coefficient, De, and a cytoplasmic diffusion coefficient, Dcyt, have been measured among the developing epidermal cells of a larval beetle, Tenebrio molitor L., to determine the contribution of the junctional channels to intercellular diffusion. Tracer diffusion was measured by injecting fluorescent tracers into cells and quantitating the rate of subsequent spread into adjacent cells. Cytoplasmic diffusion was determined by fluorescence photobleaching. These experiments show that gap junctional channels constitute approximately 70-80% of the total cell-to-cell resistance to the diffusion of organic tracers at high concentrations in this tissue. At low concentrations, however, the binding of tracer to cytoplasm slows down the cytoplasmic diffusion, which may limit intercellular diffusion.

  4. Modulation of Brain Hemichannels and Gap Junction Channels by Pro-Inflammatory Agents and Their Possible Role in Neurodegeneration

    PubMed Central

    Sáez, Pablo J.; Shoji, Kenji F.; Schalper, Kurt A.; Palacios–Prado, Nicolás; Velarde, Victoria; Giaume, Christian; Bennett, Michael V.L.; Sáez, Juan C.

    2009-01-01

    Abstract In normal brain, neurons, astrocytes, and oligodendrocytes, the most abundant and active cells express pannexins and connexins, protein subunits of two families forming membrane channels. Most available evidence indicates that in mammals endogenously expressed pannexins form only hemichannels and connexins form both gap junction channels and hemichannels. Whereas gap junction channels connect the cytoplasm of contacting cells and coordinate electric and metabolic activity, hemichannels communicate the intra- and extracellular compartments and serve as a diffusional pathway for ions and small molecules. A subthreshold stimulation by acute pathological threatening conditions (e.g., global ischemia subthreshold for cell death) enhances neuronal Cx36 and glial Cx43 hemichannel activity, favoring ATP release and generation of preconditioning. If the stimulus is sufficiently deleterious, microglia become overactivated and release bioactive molecules that increase the activity of hemichannels and reduce gap junctional communication in astroglial networks, depriving neurons of astrocytic protective functions, and further reducing neuronal viability. Continuous glial activation triggered by low levels of anomalous proteins expressed in several neurodegenerative diseases induce glial hemichannel and gap junction channel disorders similar to those of acute inflammatory responses triggered by ischemia or infectious diseases. These changes are likely to occur in diverse cell types of the CNS and contribute to neurodegeneration during inflammatory process. Antiox. Redox Signal. 11, 369–399. PMID:18816186

  5. Gap junction- and hemichannel-independent actions of connexins.

    PubMed

    Jiang, Jean X; Gu, Sumin

    2005-06-10

    Connexins have been known to be the protein building blocks of gap junctions and mediate cell-cell communication. In contrast to the conventional dogma, recent evidence suggests that in addition to forming gap junction channels, connexins possess gap junction-independent functions. One important gap junction-independent function for connexins is to serve as the major functional component for hemichannels, the un-apposed halves of gap junctions. Hemichannels, as independent functional units, play roles that are different from that of gap junctions in the cell. The other functions of connexins appear to be gap junction- and hemichannel-independent. Published studies implicate the latter functions of connexins in cell growth, differentiation, tumorigenicity, injury, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. In this review, gap junction- and hemichannel-independent functions of connexins are summarized, and the molecular mechanisms underlying these connexin functions are speculated and discussed.

  6. Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.

    PubMed

    Nakagawa, So; Gong, Xiang-Qun; Maeda, Shoji; Dong, Yuhua; Misumi, Yuko; Tsukihara, Tomitake; Bai, Donglin

    2011-06-03

    The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.

  7. Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

    PubMed

    Ahir, Bhavesh K; Pratten, Margaret K

    2014-01-01

    Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

  8. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  9. Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide‐gated channels

    PubMed Central

    Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G.; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre‐Olivier; Hell, Stefan W.

    2017-01-01

    Key points Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell‐to‐cell diffusion of ions, metabolites and second messengers.Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood–brain barrier endothelial cell line hCMEC/D3.Although the increased gap junction coupling is cAMP‐dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase.We found that cAMP activates cyclic nucleotide‐gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling.The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood–brain barrier. Abstract The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT‐PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2‐phenylaminoadenosine (2‐PAA) on the gap junction coupling. We found that 2‐PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration‐dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2‐PAA‐related enhancement of gap junction coupling. In contrast, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2‐PAA‐related enhancement of gap junction coupling. Moreover, we observed a 2

  10. Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

    PubMed

    Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Hell, Stefan W; Ngezahayo, Anaclet

    2017-04-15

    Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A 2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca 2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A 2A and A 2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca 2+ with BAPTA, or the absence of external Ca 2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of

  11. Role of heteromeric gap junctions in the cytotoxicity of cisplatin.

    PubMed

    Tong, Xuhui; Dong, Shuying; Yu, Meiling; Wang, Qin; Tao, Liang

    2013-08-09

    In several systems, the presence of gap junctions made of a single connexin has been shown to enhance the cytotoxicity of cisplatin. However, most gap junction channels in vivo appear to be heteromeric (composed of more than one connexin isoform). Here we explore in HeLa cells the cytotoxicity to cisplatin that is enhanced by heteromeric gap junctions composed of Cx26 and Cx32, which have been shown to be more selective among biological permeants than the corresponding homomeric channels. We found that survival and subsequent proliferation of cells exposed to cisplatin were substantially reduced when gap junctions were present than when there were no gap junctions. Functional inhibition of gap junctions by oleamide enhanced survival/proliferation, and enhancement of gap junctions by retinoic acid decreased survival/proliferation. These effects occurred only in high density cultures, and the treatments were without effect when there was no opportunity for gap junction formation. The presence of functional gap junctions enhanced apoptosis as reflected in markers of both early-stage and late-stage apoptosis. Furthermore, analysis of caspases 3, 8 and 9 showed that functional gap junctions specifically induced apoptosis by the mitochondrial pathway. These results demonstrate that heteromeric Cx26/Cx32 gap junctions increase the cytotoxicity of cisplatin by induction of apoptosis via the mitochondrial pathway. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Gap-junction coupling and ATP-sensitive potassium channels in human β -cell clusters: Effects on emergent dynamics

    NASA Astrophysics Data System (ADS)

    Loppini, A.; Pedersen, M. G.; Braun, M.; Filippi, S.

    2017-09-01

    The importance of gap-junction coupling between β cells in pancreatic islets is well established in mouse. Such ultrastructural connections synchronize cellular activity, confine biological heterogeneity, and enhance insulin pulsatility. Dysfunction of coupling has been associated with diabetes and altered β -cell function. However, the role of gap junctions between human β cells is still largely unexplored. By using patch-clamp recordings of β cells from human donors, we previously estimated electrical properties of these channels by mathematical modeling of pairs of human β cells. In this work we revise our estimate by modeling triplet configurations and larger heterogeneous clusters. We find that a coupling conductance in the range 0.005 -0.020 nS/pF can reproduce experiments in almost all the simulated arrangements. We finally explore the consequence of gap-junction coupling of this magnitude between β cells with mutant variants of the ATP-sensitive potassium channels involved in some metabolic disorders and diabetic conditions, translating studies performed on rodents to the human case. Our results are finally discussed from the perspective of therapeutic strategies. In summary, modeling of more realistic clusters with more than two β cells slightly lowers our previous estimate of gap-junction conductance and gives rise to patterns that more closely resemble experimental traces.

  13. A history of gap junction structure: hexagonal arrays to atomic resolution.

    PubMed

    Grosely, Rosslyn; Sorgen, Paul L

    2013-02-01

    Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.

  14. Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells.

    PubMed

    Beckmann, Anja; Grissmer, Alexander; Krause, Elmar; Tschernig, Thomas; Meier, Carola

    2016-03-01

    Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.

  15. Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics.

    PubMed

    Solan, Joell L; Lampe, Paul D

    2018-01-01

    Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (<1000Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP 3 ) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes.

    PubMed

    Phelan, P; Stebbings, L A; Baines, R A; Bacon, J P; Davies, J A; Ford, C

    1998-01-08

    In most multicellular organisms direct cell-cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

  17. General anesthetics have differential inhibitory effects on gap junction channels and hemichannels in astrocytes and neurons.

    PubMed

    Liu, Xinhe; Gangoso, Ester; Yi, Chenju; Jeanson, Tiffany; Kandelman, Stanislas; Mantz, Jean; Giaume, Christian

    2016-04-01

    Astrocytes represent a major non-neuronal cell population actively involved in brain functions and pathologies. They express a large amount of gap junction proteins that allow communication between adjacent glial cells and the formation of glial networks. In addition, these membrane proteins can also operate as hemichannels, through which "gliotransmitters" are released, and thus contribute to neuroglial interaction. There are now reports demonstrating that alterations of astroglial gap junction communication and/or hemichannel activity impact neuronal and synaptic activity. Two decades ago we reported that several general anesthetics inhibited gap junctions in primary cultures of astrocytes (Mantz et al., (1993) Anesthesiology 78(5):892-901). As there are increasing studies investigating neuroglial interactions in anesthetized mice, we here updated this previous study by employing acute cortical slices and by characterizing the effects of general anesthetics on both astroglial gap junctions and hemichannels. As hemichannel activity is not detected in cortical astrocytes under basal conditions, we treated acute slices with the endotoxin LPS or proinflammatory cytokines to induce hemichannel activity in astrocytes, which in turn activated neuronal hemichannels. We studied two extensively used anesthetics, propofol and ketamine, and the more recently developed dexmedetomidine. We report that these drugs have differential inhibitory effects on gap junctional communication and hemichannel activity in astrocytes when used in their respective, clinically relevant concentrations, and that dexmedetomidine appears to be the least effective on both channel functions. In addition, the three anesthetics have similar effects on neuronal hemichannels. Altogether, our observations may contribute to optimizing the selection of anesthetics for in vivo animal studies. © 2015 Wiley Periodicals, Inc.

  18. Non-invasive microfluidic gap junction assay.

    PubMed

    Chen, Sisi; Lee, Luke P

    2010-03-01

    Gap junctions are protein channels between cells that allow direct electrical and metabolic coupling via the exchange of biomolecules and ions. Their expression, though ubiquitous in most mammalian cell types, is especially important for the proper functioning of cardiac and neuronal systems. Many existing methods for studying gap junction communication suffer from either unquantifiable data or difficulty of use. Here, we measure the extent of dye spread and effective diffusivities through gap junction connected cells using a quantitative microfluidic cell biology platform. After loading dye by hydrodynamic focusing of calcein/AM, dye transfer dynamics into neighboring, unexposed cells can be monitored via timelapse fluorescent microscopy. By using a selective microfluidic dye loading over a confluent layer of cells, we found that high expression of gap junctions in C6 cells transmits calcein across the monolayer with an effective diffusivity of 3.4 x 10(-13) m(2)/s, which are highly coupled by Cx43. We also found that the gap junction blocker 18alpha-GA works poorly in the presence of serum even at high concentrations (50 microM); however, it is highly effective down to 2.5 microM in the absence of serum. Furthermore, when the drug is washed out, dye spread resumes rapidly within 1 min for all doses, indicating the drug does not affect transcriptional regulation of connexins in these Cx43+ cells, in contrast to previous studies. This integrated microfluidic platform enables the in situ monitoring of gap junction communication, yielding dynamic information about intercellular molecular transfer and pharmacological inhibition and recovery.

  19. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

    PubMed

    Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  20. Gap junctions in Malpighian tubules of Aedes aegypti.

    PubMed

    Weng, Xing-He; Piermarini, Peter M; Yamahiro, Atsuko; Yu, Ming-Jiun; Aneshansley, Daniel J; Beyenbach, Klaus W

    2008-02-01

    We present electrical, physiological and molecular evidence for substantial electrical coupling of epithelial cells in Malpighian tubules via gap junctions. Current was injected into one principal cell of the isolated Malpighian tubule and membrane voltage deflections were measured in that cell and in two neighboring principal cells. By short-circuiting the transepithelial voltage with the diuretic peptide leucokinin-VIII we largely eliminated electrical coupling of principal cells through the tubule lumen, thereby allowing coupling through gap junctions to be analyzed. The analysis of an equivalent electrical circuit of the tubule yielded an average gap-junction resistance (R(gj)) of 431 kOmega between two cells. This resistance would stem from 6190 open gap-junctional channels, assuming the high single gap-junction conductance of 375 pS found in vertebrate tissues. The addition of the calcium ionophore A23187 (2 micromol l(-1)) to the peritubular Ringer bath containing 1.7 mmol l(-1) Ca(2+) did not affect the gap-junction resistance, but metabolic inhibition of the tubule with dinitrophenol (0.5 mmol l(-1)) increased the gap-junction resistance 66-fold, suggesting the regulation of gap junctions by ATP. Lucifer Yellow injected into a principal cell did not appear in neighboring principal cells. Thus, gap junctions allow the passage of current but not Lucifer Yellow. Using RT-PCR we found evidence for the expression of innexins 1, 2, 3 and 7 (named after their homologues in Drosophila) in Malpighian tubules. The physiological demonstration of gap junctions and the molecular evidence for innexin in Malpighian tubules of Aedes aegypti call for the double cable model of the tubule, which will improve the measurement and the interpretation of electrophysiological data collected from Malpighian tubules.

  1. Functional asymmetry and plasticity of electrical synapses interconnecting neurons through a 36-state model of gap junction channel gating

    PubMed Central

    Kraujalis, Tadas; Maciunas, Kestutis

    2017-01-01

    We combined the Hodgkin–Huxley equations and a 36-state model of gap junction channel gating to simulate electrical signal transfer through electrical synapses. Differently from most previous studies, our model can account for dynamic modulation of junctional conductance during the spread of electrical signal between coupled neurons. The model of electrical synapse is based on electrical properties of the gap junction channel encompassing two fast and two slow gates triggered by the transjunctional voltage. We quantified the influence of a difference in input resistances of electrically coupled neurons and instantaneous conductance–voltage rectification of gap junctions on an asymmetry of cell-to-cell signaling. We demonstrated that such asymmetry strongly depends on junctional conductance and can lead to the unidirectional transfer of action potentials. The simulation results also revealed that voltage spikes, which develop between neighboring cells during the spread of action potentials, can induce a rapid decay of junctional conductance, thus demonstrating spiking activity-dependent short-term plasticity of electrical synapses. This conclusion was supported by experimental data obtained in HeLa cells transfected with connexin45, which is among connexin isoforms expressed in neurons. Moreover, the model allowed us to replicate the kinetics of junctional conductance under different levels of intracellular concentration of free magnesium ([Mg2+]i), which was experimentally recorded in cells expressing connexin36, a major neuronal connexin. We demonstrated that such [Mg2+]i-dependent long-term plasticity of the electrical synapse can be adequately reproduced through the changes of slow gate parameters of the 36-state model. This suggests that some types of chemical modulation of gap junctions can be executed through the underlying mechanisms of voltage gating. Overall, the developed model accounts for direction-dependent asymmetry, as well as for short- and long

  2. Gap junctions modulate glioma invasion by direct transfer of microRNA.

    PubMed

    Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L; Naus, Christian C

    2015-06-20

    The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

  3. Gap junctions modulate glioma invasion by direct transfer of microRNA

    PubMed Central

    Hong, Xiaoting; Sin, Wun Chey; Harris, Andrew L.; Naus, Christian C.

    2015-01-01

    The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity. PMID:25978028

  4. Oligomeric structure and functional characterization of Caenorhabditis elegans Innexin-6 gap junction protein.

    PubMed

    Oshima, Atsunori; Matsuzawa, Tomohiro; Nishikawa, Kouki; Fujiyoshi, Yoshinori

    2013-04-12

    Innexin is the molecular component of invertebrate gap junctions. Here we successfully expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels and characterized the molecular dimensions and channel permeability using electron microscopy (EM) and microinjection of fluorescent dye tracers, respectively. Negative staining and thin-section EM of isolated INX-6 gap junction membranes revealed a loosely packed hexagonal lattice and a greater cross-sectional width than that of connexin26 and connexin43 (Cx43)-GFP. In gel filtration analysis, the elution profile of purified INX-6 channels in dodecyl maltoside solution exhibited a peak at ∼400 kDa that was shifted to ∼800 kDa in octyl glucose neopentyl glycol. We also obtained the class averages of purified INX-6 channels from these peak fractions by single particle analysis. The class average from the ∼800-kDa fraction showed features of the junction form with a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class averages from the ∼400-kDa fraction showed diameters of up to 140 Å in the presence of detergent micelles. These findings indicate that the purified INX-6 channels are predominantly hemichannels in dodecyl maltoside and docked junction channels in octyl glucose neopentyl glycol. Dye transfer experiments revealed that the INX-6-GFP-His channels are permeable to 3- and 10-kDa tracers, whereas no significant amounts of these tracers passed through the Cx43-GFP channels. Based on these findings, INX-6 channels have a larger overall structure and greater permeability than connexin channels.

  5. Increase of gap junction activities in SW480 human colorectal cancer cells.

    PubMed

    Bigelow, Kristina; Nguyen, Thu A

    2014-07-09

    Colorectal cancer is one of the most common cancers in the United States with an early detection rate of only 39%. Colorectal cancer cells along with other cancer cells exhibit many deficiencies in cell-to-cell communication, particularly gap junctional intercellular communication (GJIC). GJIC has been reported to diminish as cancer cells progress. Gap junctions are intercellular channels composed of connexin proteins, which mediate the direct passage of small molecules from one cell to the next. They are involved in the regulation of the cell cycle, cell differentiation, and cell signaling. Since the regulation of gap junctions is lost in colorectal cancer cells, the goal of this study is to determine the effect of GJIC restoration in colorectal cancer cells. Gap Junction Activity Assay and protein analysis were performed to evaluate the effects of overexpression of connexin 43 (Cx43) and treatment of PQ1, a small molecule, on GJIC. Overexpression of Cx43 in SW480 colorectal cancer cells causes a 6-fold increase of gap junction activity compared to control. This suggests that overexpressing Cx43 can restore GJIC. Furthermore, small molecule like PQ1 directly targeting gap junction channel was used to increase GJIC. Gap junction enhancers, PQ1, at 200 nM showed a 4-fold increase of gap junction activity in SW480 cells. A shift from the P0 to the P2 isoform of Cx43 was seen after 1 hour treatment with 200 nM PQ1. Overexpression of Cx43 and treatment of PQ1 can directly increase gap junction activity. The findings provide an important implication in which restoration of gap junction activity can be targeted for drug development.

  6. Astrocyte dysfunction in temporal lobe epilepsy: K+ channels and gap junction coupling.

    PubMed

    Steinhäuser, Christian; Seifert, Gerald; Bedner, Peter

    2012-08-01

    Astrocytes are endowed with the machinery to sense and respond to neuronal activity. Recent work has demonstrated that astrocytes play important physiological roles in the CNS, e.g., they synchronize action potential firing, ensure ion homeostasis, transmitter clearance and glucose metabolism, and regulate the vascular tone. Astrocytes are abundantly coupled through gap junctions, which is a prerequisite to redistribute elevated K(+) from sites of excessive neuronal activity to sites of lower extracellular K(+) concentration. Recent studies identified dysfunctional astrocytes as crucial players in epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization, and function of astroglial inwardly rectifying K(+) (Kir) channels, particularly Kir4.1, which is suspected to entail impaired K(+) buffering. Gap junctions in astrocytes appear to play a dual role: on the one hand they counteract the generation of hyperactivity by facilitating clearance of elevated extracellular K(+) levels while in contrast, they constitute a pathway for energetic substrate delivery to fuel neuronal (hyper)activity. Recent work suggests that astrocyte dysfunction is causative of the generation or spread of seizure activity. Thus, astrocytes should be considered as promising targets for alternative antiepileptic therapies. Copyright © 2012 Wiley Periodicals, Inc.

  7. Gap junctions in cells of the immune system: structure, regulation and possible functional roles.

    PubMed

    Sáez, J C; Brañes, M C; Corvalán, L A; Eugenín, E A; González, H; Martínez, A D; Palisson, F

    2000-04-01

    Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family. According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps of the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response of the immune system.

  8. Oligomeric Structure and Functional Characterization of Caenorhabditis elegans Innexin-6 Gap Junction Protein*

    PubMed Central

    Oshima, Atsunori; Matsuzawa, Tomohiro; Nishikawa, Kouki; Fujiyoshi, Yoshinori

    2013-01-01

    Innexin is the molecular component of invertebrate gap junctions. Here we successfully expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels and characterized the molecular dimensions and channel permeability using electron microscopy (EM) and microinjection of fluorescent dye tracers, respectively. Negative staining and thin-section EM of isolated INX-6 gap junction membranes revealed a loosely packed hexagonal lattice and a greater cross-sectional width than that of connexin26 and connexin43 (Cx43)-GFP. In gel filtration analysis, the elution profile of purified INX-6 channels in dodecyl maltoside solution exhibited a peak at ∼400 kDa that was shifted to ∼800 kDa in octyl glucose neopentyl glycol. We also obtained the class averages of purified INX-6 channels from these peak fractions by single particle analysis. The class average from the ∼800-kDa fraction showed features of the junction form with a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class averages from the ∼400-kDa fraction showed diameters of up to 140 Å in the presence of detergent micelles. These findings indicate that the purified INX-6 channels are predominantly hemichannels in dodecyl maltoside and docked junction channels in octyl glucose neopentyl glycol. Dye transfer experiments revealed that the INX-6-GFP-His channels are permeable to 3- and 10-kDa tracers, whereas no significant amounts of these tracers passed through the Cx43-GFP channels. Based on these findings, INX-6 channels have a larger overall structure and greater permeability than connexin channels. PMID:23460640

  9. Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

    PubMed

    Eckardt, D; Theis, M; Degen, J; Ott, T; van Rijen, H V M; Kirchhoff, S; Kim, J-S; de Bakker, J M T; Willecke, K

    2004-01-01

    The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

  10. Desipramine prevents cardiac gap junction uncoupling.

    PubMed

    Jozwiak, Joanna; Dietze, Anna; Grover, Rajiv; Savtschenko, Alex; Etz, Christian; Mohr, Friedrich W; Dhein, Stefan

    2012-11-01

    Uncoupling of cardiac gap junction channels is an important arrhythmogenic mechanism in ischemia/reperfusion. Antiarrhythmic peptide AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)) has been shown to prevent acidosis-induced uncoupling and ischemia-related increase in dispersion. Previous structure-effect investigations and subsequent computer modeling studies indicated that the tricyclic antidepressant desipramine may exert similar effects as AAP10. We assessed the binding of (14)C-AAP10 to membranes of rabbit cardiac ventricles and its displacement with desipramine in a classical radioligand binding and competition study. Gap junction currents were measured between isolated pairs of human atrial cardiomyocytes under normal and acidotic (pH 6.3) conditions with or without 1 μmol/l desipramine using dual whole-cell voltage clamp. The effect of 1 μmol/l desipramine was assessed in isolated rabbit hearts (Langendorff technique) undergoing local ischemia by coronary occlusion with 256-channel electrophysiological mapping and subsequent analysis of connexin43 (Cx43) expression, phosphorylation (Western blot), and subcellular localization (immunohistology). We found saturable (14)C-AAP10 binding to cardiac membranes (K (D), 0.29 ± 0.11 nmol/l; B (max), 42.5 ± 7.2 pmol/mg) which could be displaced by desipramine with a K (D.High) = 0.14 μmol/l and a K (D.Low) = 22 μmol/l. Acidosis reduced the gap junction conductance in human cardiomyocyte pairs from 24.1 ± 4.7 to 11.5 ± 2.5 nS, which could be significantly reversed by desipramine (26.6 ± 4.8 nS). In isolated hearts, ischemia resulted in significantly increased dispersion of activation-recovery intervals, loss of membrane Cx43, and dephosphorylation of Cx43, which all could be prevented by desipramine. Desipramine seems to prevent the uncoupling of cardiac gap junctions and ischemia-related increase in dispersion.

  11. Structural basis for the selective permeability of channels made of communicating junction proteins

    PubMed Central

    Ek-Vitorin, Jose F.; Burt, Janis M.

    2012-01-01

    The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore’s limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. PMID:22342665

  12. Physiological and physiopathological aspects of connexins and communicating gap junctions in spermatogenesis

    PubMed Central

    Pointis, Georges; Gilleron, Jérome; Carette, Diane; Segretain, Dominique

    2010-01-01

    Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell–cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli–Sertoli cell functional synchronization and the Sertoli–germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation. PMID:20403873

  13. Structural basis for the selective permeability of channels made of communicating junction proteins.

    PubMed

    Ek-Vitorin, Jose F; Burt, Janis M

    2013-01-01

    The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore's limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

    PubMed

    Smendziuk, Christopher M; Messenberg, Anat; Vogl, A Wayne; Tanentzapf, Guy

    2015-08-01

    Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues. © 2015. Published by The Company of Biologists Ltd.

  15. Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions.

    PubMed

    Musa, Hassan; Fenn, Edward; Crye, Mark; Gemel, Joanna; Beyer, Eric C; Veenstra, Richard D

    2004-06-15

    Connexin40 (Cx40) contains a specific binding site for spermine (affinity approximately 100 microm) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mm spermine, reduced the transjunctional voltage (V(j)) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (G(min)) and V(j) gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V(j) sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for V(j)-dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.

  16. Regulation of osteoclastogenesis by gap junction communication.

    PubMed

    Matemba, Stephen F; Lie, Anita; Ransjö, Maria

    2006-10-01

    Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant > or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures stimulated by parathyroid hormone (PTH; 10 nM) or forskolin (FSK; 1 microM). Furthermore, CBX (100 microM) significantly inhibited prostaglandin E2 (PGE2; 10 microM) and 1,25(OH)2-vitamin D3 stimulated osteoclast differentiation in the mouse bone marrow cultures. Consequently, quantitative real-time polymerase chain reaction (PCR) analysis demonstrated that CBX downregulated the expression of osteoclast phenotypic markers, but without having any significant effects on RANK, RANKL, and osteoprotegerin (OPG) mRNA expression. However, the results demonstrated that CBX significantly inhibits RANKL-stimulated (100 ng/ml) osteoclastogenesis in the mouse bone marrow cultures. Taken together, our results suggests that gap junctional diffusion of messenger molecules interacts with signaling pathways downstream RANKL in osteoclast differentiation. Further studies are required to define the precise mechanisms and molecular targets involved. Copyright 2006 Wiley-Liss, Inc.

  17. Gap state charge induced spin-dependent negative differential resistance in tunnel junctions

    NASA Astrophysics Data System (ADS)

    Jiang, Jun; Zhang, X.-G.; Han, X. F.

    2016-04-01

    We propose and demonstrate through first-principles calculation a new spin-dependent negative differential resistance (NDR) mechanism in magnetic tunnel junctions (MTJ) with cubic cation disordered crystals (CCDC) AlO x or Mg1-x Al x O as barrier materials. The CCDC is a class of insulators whose band gap can be changed by cation doping. The gap becomes arched in an ultrathin layer due to the space charge formed from metal-induced gap states. With an appropriate combination of an arched gap and a bias voltage, NDR can be produced in either spin channel. This mechanism is applicable to 2D and 3D ultrathin junctions with a sufficiently small band gap that forms a large space charge. It provides a new way of controlling the spin-dependent transport in spintronic devices by an electric field. A generalized Simmons formula for tunneling current through junction with an arched gap is derived to show the general conditions under which ultrathin junctions may exhibit NDR.

  18. Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly

    PubMed Central

    1994-01-01

    The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level. PMID:7806568

  19. Monitoring gap junctional communication in astrocytes from acute adult mouse brain slices using the gap-FRAP technique.

    PubMed

    Yi, Chenju; Teillon, Jérémy; Koulakoff, Annette; Berry, Hugues; Giaume, Christian

    2018-06-01

    Intercellular communication through gap junction channels plays a key role in cellular homeostasis and in synchronizing physiological functions, a feature that is modified in number of pathological situations. In the brain, astrocytes are the cell population that expresses the highest amount of gap junction proteins, named connexins. Several techniques have been used to assess the level of gap junctional communication in astrocytes, but so far they remain very difficult to apply in adult brain tissue. Here, using specific loading of astrocytes with sulforhodamine 101, we adapted the gap-FRAP (Fluorescence Recovery After Photobleaching) to acute hippocampal slices from 9 month-old adult mice. We show that gap junctional communication monitored in astrocytes with this technique was inhibited either by pharmacological treatment with a gap junctional blocker or in mice lacking the two main astroglial connexins, while a partial inhibition was measured when only one connexin was knocked-out. We validate this approach using a mathematical model of sulforhodamine 101 diffusion in an elementary astroglial network and a quantitative analysis of the exponential fits to the fluorescence recovery curves. Consequently, we consider that the adaptation of the gap-FRAP technique to acute brain slices from adult mice provides an easy going and valuable approach that allows overpassing this age-dependent obstacle and will facilitate the investigation of gap junctional communication in adult healthy or pathological brain. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Smad ubiquitination regulatory factor-2 controls gap junction intercellular communication by modulating endocytosis and degradation of connexin43.

    PubMed

    Fykerud, Tone Aase; Kjenseth, Ane; Schink, Kay Oliver; Sirnes, Solveig; Bruun, Jarle; Omori, Yasufumi; Brech, Andreas; Rivedal, Edgar; Leithe, Edward

    2012-09-01

    Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junction channels are made of a family of integral membrane proteins called connexins, of which the best-studied member is connexin43. Gap junctions are dynamic plasma membrane domains, and connexin43 has a high turnover rate in most tissue types. However, the mechanisms involved in the regulation of connexin43 endocytosis and transport to lysosomes are still poorly understood. Here, we demonstrate by live-cell imaging analysis that treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) induces endocytosis of subdomains of connexin43 gap junctions. The internalized, connexin43-enriched vesicles were found to fuse with early endosomes, which was followed by transport of connexin43 to the lumen of early endosomes. The HECT E3 ubiquitin ligase smad ubiquitination regulatory factor-2 (Smurf2) was found to be recruited to connexin43 gap junctions in response to TPA treatment. Depletion of Smurf2 by small interfering RNA resulted in enhanced levels of connexin43 gap junctions between adjacent cells and increased gap junction intercellular communication. Smurf2 depletion also counteracted the TPA-induced endocytosis and degradation of connexin43. Collectively, these data identify Smurf2 as a novel regulator of connexin43 gap junctions.

  1. Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

    PubMed Central

    Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; White, Thomas W.; Brink, Peter R.

    2011-01-01

    We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium. PMID:21624945

  2. Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

    PubMed

    Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

    2016-01-01

    Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.

  3. Regulation of Gap Junction Dynamics by UNC-44/ankyrin and UNC-33/CRMP through VAB-8 in C. elegans Neurons

    PubMed Central

    Yan, Dong

    2016-01-01

    Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions. PMID:27015090

  4. Inhibition of gap junction currents by the abused solvent toluene.

    PubMed

    Del Re, Angelo M; Woodward, John J

    2005-05-09

    Abused inhalants are a large class of compounds that are inhaled for their intoxicating and mood altering effects. They include chemicals with known therapeutic uses such as anesthetic gases as well as volatile organic solvents like toluene that are found in paint thinners and adhesives. Because of their widespread commercial use and availability, inhalants are often among the first drugs that children encounter and use of these compounds is often associated with adverse acute and long-term consequences. The cellular and molecular sites of action for abused inhalants is not well known although recent studies report that toluene and other organic solvents alter the activity of specific ligand- and voltage-gated ion channels that regulate cellular excitability. As part of an ongoing effort to define molecular sites of action for abused inhalants, this study examined the effect of toluene on the function of gap junction proteins endogenously expressed in human embryonic kidney (HEK 293) cells. Gap junctions allow cell-to-cell electrical communication as well as passage of small molecular weight substances and are critical for synchronizing cellular activity in certain tissues. Gap junction currents in HEK 293 cells were measured during brief voltage steps using patch-clamp electrophysiology and were blocked by known gap junction blockers confirming expression of connexin proteins in these cells. Toluene dose-dependently inhibited these conductances with threshold effects appearing at approximately 0.4 mM and near complete inhibition occurring at concentrations of 1 mM and higher. The estimated EC50 value for toluene inhibition of gap junction currents in HEK 293 cells was 0.57 mM. The results of these studies suggest that volatile solvents including toluene may produce some of their effects by disrupting inter-cellular communication mediated by gap junction proteins.

  5. Establishment of the Dual Whole Cell Recording Patch Clamp Configuration for the Measurement of Gap Junction Conductance.

    PubMed

    Veenstra, Richard D

    2016-01-01

    The development of the patch clamp technique has enabled investigators to directly measure gap junction conductance between isolated pairs of small cells with resolution to the single channel level. The dual patch clamp recording technique requires specialized equipment and the acquired skill to reliably establish gigaohm seals and the whole cell recording configuration with high efficiency. This chapter describes the equipment needed and methods required to achieve accurate measurement of macroscopic and single gap junction channel conductances. Inherent limitations with the dual whole cell recording technique and methods to correct for series access resistance errors are defined as well as basic procedures to determine the essential electrical parameters necessary to evaluate the accuracy of gap junction conductance measurements using this approach.

  6. Abundance of gap junctions at glutamatergic mixed synapses in adult Mosquitofish spinal cord neurons

    PubMed Central

    Serrano-Velez, Jose L.; Rodriguez-Alvarado, Melanie; Torres-Vazquez, Irma I.; Fraser, Scott E.; Yasumura, Thomas; Vanderpool, Kimberly G.; Rash, John E.; Rosa-Molinar, Eduardo

    2014-01-01

    “Dye-coupling”, whole-mount immunohistochemistry for gap junction channel protein connexin 35 (Cx35), and freeze-fracture replica immunogold labeling (FRIL) reveal an abundance of electrical synapses/gap junctions at glutamatergic mixed synapses in the 14th spinal segment that innervates the adult male gonopodium of Western Mosquitofish, Gambusia affinis (Mosquitofish). To study gap junctions’ role in fast motor behavior, we used a minimally-invasive neural-tract-tracing technique to introduce gap junction-permeant or -impermeant dyes into deep muscles controlling the gonopodium of the adult male Mosquitofish, a teleost fish that rapidly transfers (complete in <20 mS) spermatozeugmata into the female reproductive tract. Dye-coupling in the 14th spinal segment controlling the gonopodium reveals coupling between motor neurons and a commissural primary ascending interneuron (CoPA IN) and shows that the 14th segment has an extensive and elaborate dendritic arbor and more gap junctions than do other segments. Whole-mount immunohistochemistry for Cx35 results confirm dye-coupling and show it occurs via gap junctions. Finally, FRIL shows that gap junctions are at mixed synapses and reveals that >50 of the 62 gap junctions at mixed synapses are in the 14th spinal segment. Our results support and extend studies showing gap junctions at mixed synapses in spinal cord segments involved in control of genital reflexes in rodents, and they suggest a link between mixed synapses and fast motor behavior. The findings provide a basis for studies of specific roles of spinal neurons in the generation/regulation of sex-specific behavior and for studies of gap junctions’ role in regulating fast motor behavior. Finally, the CoPA IN provides a novel candidate neuron for future studies of gap junctions and neural control of fast motor behaviors. PMID:25018700

  7. Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions

    PubMed Central

    Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A.; Bukauskas, Feliksas F.

    2018-01-01

    Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions. PMID:29706896

  8. Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

    PubMed

    Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

    2018-01-01

    Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

  9. Relating proton pumps with gap junctions: colocalization of ductin, the channel-forming subunit c of V-ATPase, with subunit a and with innexins 2 and 3 during Drosophila oogenesis.

    PubMed

    Lautemann, Julia; Bohrmann, Johannes

    2016-07-13

    Ion-transport mechanisms and gap junctions are known to cooperate in creating bioelectric phenomena, like pH gradients, voltage gradients and ion fluxes within single cells, tissues, organs, and whole organisms. Such phenomena have been shown to play regulatory roles in a variety of developmental and regenerative processes. Using Drosophila oogenesis as a model system, we aim at characterizing in detail the mechanisms underlying bioelectric phenomena in order to reveal their regulatory functions. We, therefore, investigated the stage-specific distribution patterns of V-ATPase components in relation to gap-junction proteins. We analysed the localization of the V-ATPase components ductin (subunit c) and subunit a, and the gap-junction components innexins 2 and 3, especially in polar cells, border cells, stalk cells and centripetally migrating cells. These types of follicle cells had previously been shown to exhibit characteristic patterns of membrane channels as well as membrane potential and intracellular pH. Stage-specifically, ductin and subunit a were found either colocalized or separately enriched in different regions of soma and germ-line cells. While ductin was often more prominent in plasma membranes, subunit a was more prominent in cytoplasmic and nuclear vesicles. Particularly, ductin was enriched in polar cells, stalk cells, and nurse-cell membranes, whereas subunit a was enriched in the cytoplasm of border cells, columnar follicle cells and germ-line cells. Comparably, ductin and both innexins 2 and 3 were either colocalized or separately enriched in different cellular regions. While ductin often showed a continuous membrane distribution, the distribution of both innexins was mostly punctate. Particularly, ductin was enriched in polar cells and stalk cells, whereas innexin 2 was enriched in the oolemma, and innexin 3 in centripetally migrating follicle cells. In lateral follicle-cell membranes, the three proteins were found colocalized as well as

  10. Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer

    PubMed Central

    Jin, Xin; Valiente, Manuel; Er, Ekrem Emrah; Lopez-Soto, Alejandro; Jacob, Leni; Patwa, Ruzeen; Shah, Hardik; Xu, Ke; Cross, Justin R.; Massagué, Joan

    2016-01-01

    SUMMARY Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. Breast and lung cancer cells express protocadherin 7 (PCDH7) to favor the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells employ these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines IFNα and TNFα. As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, which support tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle for the applicability of this therapeutic strategy to treat established brain metastasis. PMID:27225120

  11. Improving cardiac gap junction communication as a new antiarrhythmic mechanism: the action of antiarrhythmic peptides.

    PubMed

    Dhein, Stefan; Hagen, Anja; Jozwiak, Joanna; Dietze, Anna; Garbade, Jens; Barten, Markus; Kostelka, Martin; Mohr, Friedrich-Wilhelm

    2010-03-01

    Co-ordinated electrical activation of the heart is maintained by intercellular coupling of cardiomyocytes via gap junctional channels located in the intercalated disks. These channels consist of two hexameric hemichannels, docked to each other, provided by either of the adjacent cells. Thus, a complete gap junction channel is made from 12 protein subunits, the connexins. While 21 isoforms of connexins are presently known, cardiomyocytes typically are coupled by Cx43 (most abundant), Cx40 or Cx45. Some years ago, antiarrhythmic peptides were discovered and synthesised, which were shown to increase macroscopic gap junction conductance (electrical coupling) and enhance dye transfer (metabolic coupling). The lead substance of these peptides is AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-CONH(2)), a peptide with a horseshoe-like spatial structure as became evident from two-dimensional nuclear magnetic resonance studies. A stable D: -amino-acid derivative of AAP10, rotigaptide, as well as a non-peptide analogue, gap-134, has been developed in recent years. Antiarrhythmic peptides act on Cx43 and Cx45 gap junctions but not on Cx40 channels. AAP10 has been shown to enhance intercellular communication in rat, rabbit and human cardiomyocytes. Antiarrhythmic peptides are effective against ventricular tachyarrhythmias, such as late ischaemic (type IB) ventricular fibrillation, CaCl(2) or aconitine-induced arrhythmia. Interestingly, the effect of antiarrhythmic peptides is higher in partially uncoupled cells and was shown to be related to maintained Cx43 phosphorylation, while arrhythmogenic conditions like ischaemia result in Cx43 dephosphorylation and intercellular decoupling. It is still a matter of debate whether these drugs also act against atrial fibrillation. The present review outlines the development of this group of peptides and derivatives, their mode of action and molecular mechanisms, and discusses their possible therapeutic potential.

  12. Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

    PubMed

    Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

    2016-11-01

    Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

  13. Mouse rods signal through gap junctions with cones

    PubMed Central

    Asteriti, Sabrina; Gargini, Claudia; Cangiano, Lorenzo

    2014-01-01

    Rod and cone photoreceptors are coupled by gap junctions (GJs), relatively large channels able to mediate both electrical and molecular communication. Despite their critical location in our visual system and evidence that they are dynamically gated for dark/light adaptation, the full impact that rod–cone GJs can have on cone function is not known. We recorded the photovoltage of mouse cones and found that the initial level of rod input increased spontaneously after obtaining intracellular access. This process allowed us to explore the underlying coupling capacity to rods, revealing that fully coupled cones acquire a striking rod-like phenotype. Calcium, a candidate mediator of the coupling process, does not appear to be involved on the cone side of the junctional channels. Our findings show that the anatomical substrate is adequate for rod–cone coupling to play an important role in vision and, possibly, in biochemical signaling among photoreceptors. DOI: http://dx.doi.org/10.7554/eLife.01386.001 PMID:24399457

  14. Mouse rods signal through gap junctions with cones.

    PubMed

    Asteriti, Sabrina; Gargini, Claudia; Cangiano, Lorenzo

    2014-01-01

    Rod and cone photoreceptors are coupled by gap junctions (GJs), relatively large channels able to mediate both electrical and molecular communication. Despite their critical location in our visual system and evidence that they are dynamically gated for dark/light adaptation, the full impact that rod-cone GJs can have on cone function is not known. We recorded the photovoltage of mouse cones and found that the initial level of rod input increased spontaneously after obtaining intracellular access. This process allowed us to explore the underlying coupling capacity to rods, revealing that fully coupled cones acquire a striking rod-like phenotype. Calcium, a candidate mediator of the coupling process, does not appear to be involved on the cone side of the junctional channels. Our findings show that the anatomical substrate is adequate for rod-cone coupling to play an important role in vision and, possibly, in biochemical signaling among photoreceptors. DOI: http://dx.doi.org/10.7554/eLife.01386.001.

  15. Gap junction-mediated intercellular communication in the immune system.

    PubMed

    Neijssen, Joost; Pang, Baoxu; Neefjes, Jacques

    2007-01-01

    Immune cells are usually considered non-attached blood cells, which would exclude the formation of gap junctions. This is a misconception since many immune cells express connexin 43 (Cx43) and other connexins and are often residing in tissue. The role of gap junctions is largely ignored by immunologists as is the immune system in the field of gap junction research. Here, the current knowledge of the distribution of connexins and the function of gap junctions in the immune system is discussed. Gap junctions appear to play many roles in antibody productions and specific immune responses and may be important in sensing danger in tissue by the immune system. Gap junctions not only transfer electrical and metabolical but also immunological information in the form of peptides for a process called cross-presentation. This is essential for proper immune responses to viruses and possibly tumours. Until now only 40 research papers on gap junctions in the immune system appeared and this will almost certainly expand with the increased mutual interest between the fields of immunology and gap junction research.

  16. Cut-loading: a useful tool for examining the extent of gap junction tracer coupling between retinal neurons.

    PubMed

    Choi, Hee Joo; Ribelayga, Christophe P; Mangel, Stuart C

    2012-01-12

    In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent(1,2). In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue. Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions(3,4). Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling(3-8). For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation(7,8). However, not only are these studies extremely difficult to perform on

  17. Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer.

    PubMed

    Chen, Qing; Boire, Adrienne; Jin, Xin; Valiente, Manuel; Er, Ekrem Emrah; Lopez-Soto, Alejandro; Jacob, Leni; Patwa, Ruzeen; Shah, Hardik; Xu, Ke; Cross, Justin R; Massagué, Joan

    2016-05-26

    Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. We show that human and mouse breast and lung cancer cells express protocadherin 7 (PCDH7), which promotes the assembly of carcinoma-astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells use these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines such as interferon-α (IFNα) and tumour necrosis factor (TNF). As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, thereby supporting tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle that these drugs could be used to treat established brain metastasis.

  18. The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

    PubMed Central

    Guan, Xiaojun; Cravatt, Benjamin F.; Ehring, George R.; Hall, James E.; Boger, Dale L.; Lerner, Richard A.; Gilula, Norton B.

    1997-01-01

    Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction. PMID:9412472

  19. Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons.

    PubMed

    Rash, J E; Yasumura, T; Dudek, F E; Nagy, J I

    2001-03-15

    The transmembrane connexin proteins of gap junctions link extracellularly to form channels for cell-to-cell exchange of ions and small molecules. Two primary hypotheses of gap junction coupling in the CNS are the following: (1) generalized coupling occurs between neurons and glia, with some connexins expressed in both neurons and glia, and (2) intercellular junctional coupling is restricted to specific coupling partners, with different connexins expressed in each cell type. There is consensus that gap junctions link neurons to neurons and astrocytes to oligodendrocytes, ependymocytes, and other astrocytes. However, unresolved are the existence and degree to which gap junctions occur between oligodendrocytes, between oligodendrocytes and neurons, and between astrocytes and neurons. Using light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling of adult rat CNS, we investigated whether four of the best-characterized CNS connexins are each present in one or more cell types, whether oligodendrocytes also share gap junctions with other oligodendrocytes or with neurons, and whether astrocytes share gap junctions with neurons. Connexin32 (Cx32) was found only in gap junctions of oligodendrocyte plasma membranes, Cx30 and Cx43 were found only in astrocyte membranes, and Cx36 was only in neurons. Oligodendrocytes shared intercellular gap junctions only with astrocytes, with each oligodendrocyte isolated from other oligodendrocytes except via astrocyte intermediaries. Finally, neurons shared gap junctions only with other neurons and not with glial cells. Thus, the different cell types of the CNS express different connexins, which define separate pathways for neuronal versus glial gap junctional communication.

  20. Carbon Tetrachloride at Hepatotoxic Levels Blocks Reversibly Gap Junctions between Rat Hepatocytes

    NASA Astrophysics Data System (ADS)

    Saez, J. C.; Bennett, M. V. L.; Spray, D. C.

    1987-05-01

    Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent β -mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

  1. Spinal gap junctions: potential involvement in pain facilitation.

    PubMed

    Spataro, Leah E; Sloane, Evan M; Milligan, Erin D; Wieseler-Frank, Julie; Schoeniger, Diana; Jekich, Brian M; Barrientos, Ruth M; Maier, Steven F; Watkins, Linda R

    2004-09-01

    Glia are now recognized as important contributors in pathological pain creation and maintenance. Spinal cord glia exhibit extensive gap junctional connectivity, raising the possibility that glia are involved in the contralateral spread of excitation resulting in mirror image pain. In the present experiments, the gap junction decoupler carbenoxolone was administered intrathecally after induction of neuropathic pain in response to sciatic nerve inflammation (sciatic inflammatory neuropathy) or partial nerve injury (chronic constriction injury). In both neuropathic pain models, a low dose of carbenoxolone reversed mirror image mechanical allodynia, while leaving ipsilateral mechanical allodynia unaffected. Ipsilateral thermal hyperalgesia was briefly attenuated. Critically, blockade of mechanical allodynia and thermal hyperalgesia was not observed in response to intrathecal glycyrrhizic acid, a compound similar to carbenoxolone in all respects but it does not decouple gap junctions. Thus, blockade of mechanical allodynia and thermal hyperalgesia by carbenoxolone does appear to reflect an effect on gap junctions. Examination of carbenoxolone's effects on intrathecal human immunodeficiency virus type 1 gp120 showed that blockade of pain facilitation might result, at least in part, via suppression of interleukin-1 and, in turn, interleukin-6. These data provide the first suggestion that spread of excitation via gap junctions might contribute importantly to inflammatory and traumatic neuropathic pain. The current studies provide evidence for involvement of gap junctions in spinal cord pain facilitation. Intrathecal carbenoxolone, a gap junction decoupler, reversed neuropathy-induced mirror image pain and intrathecal gp120-induced allodynia. In addition, it decreased gp120-induced proinflammatory cytokines. This suggests gap junction activation might lead to proinflammatory cytokine release by distantly activated glia.

  2. Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

    PubMed Central

    Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

    2014-01-01

    The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap

  3. Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans.

    PubMed

    Farnsworth, Nikki L; Hemmati, Alireza; Pozzoli, Marina; Benninger, Richard K P

    2014-10-15

    The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose‐stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre‐diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose‐ and cAMP‐dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of

  4. Molecular mechanisms regulating formation, trafficking and processing of annular gap junctions.

    PubMed

    Falk, Matthias M; Bell, Cheryl L; Kells Andrews, Rachael M; Murray, Sandra A

    2016-05-24

    Internalization of gap junction plaques results in the formation of annular gap junction vesicles. The factors that regulate the coordinated internalization of the gap junction plaques to form annular gap junction vesicles, and the subsequent events involved in annular gap junction processing have only relatively recently been investigated in detail. However it is becoming clear that while annular gap junction vesicles have been demonstrated to be degraded by autophagosomal and endo-lysosomal pathways, they undergo a number of additional processing events. Here, we characterize the morphology of the annular gap junction vesicle and review the current knowledge of the processes involved in their formation, fission, fusion, and degradation. In addition, we address the possibility for connexin protein recycling back to the plasma membrane to contribute to gap junction formation and intercellular communication. Information on gap junction plaque removal from the plasma membrane and the subsequent processing of annular gap junction vesicles is critical to our understanding of cell-cell communication as it relates to events regulating development, cell homeostasis, unstable proliferation of cancer cells, wound healing, changes in the ischemic heart, and many other physiological and pathological cellular phenomena.

  5. Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry

    PubMed Central

    Zhong, Guoqiang; Akoum, Nazem; Appadurai, Daniel A.; Hayrapetyan, Volodya; Ahmed, Osman; Martinez, Agustin D.; Beyer, Eric C.; Moreno, Alonso P.

    2017-01-01

    In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules. PMID:28611680

  6. Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry.

    PubMed

    Zhong, Guoqiang; Akoum, Nazem; Appadurai, Daniel A; Hayrapetyan, Volodya; Ahmed, Osman; Martinez, Agustin D; Beyer, Eric C; Moreno, Alonso P

    2017-01-01

    In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2) is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj) for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge) of the crossing molecules.

  7. Pannexins and gap junction protein diversity.

    PubMed

    Shestopalov, V I; Panchin, Y

    2008-02-01

    Gap junctions (GJs) are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Connexins were initially considered to be the only proteins capable of GJ formation. Another family of GJ proteins (innexins) were first found in invertebrates and were proposed to be renamed pannexins after their orthologs were discovered in vertebrates. The lack of both connexins and pannexins in the genomes of some metazoans suggests that other, still undiscovered GJ proteins exist. In vertebrates, connexins and pannexins co-exist. Here we discuss whether vertebrate pannexins have a nonredundant role in animal physiology. Pannexin channels appear to be suited for ATP and calcium signaling and play a role in the maintenance of calcium homeostasis by mechanisms implicating both GJ and nonjunctional function. Suggested roles in the ischemic death of neurons, schizophrenia, inflammation and tumor suppression have drawn much attention to exploring the molecular properties and cellular functions of pannexins.

  8. Lovastatin inhibits gap junctional communication in cultured aortic smooth muscle cells.

    PubMed

    Shen, Jing; Wang, Li-Hong; Zheng, Liang-Rong; Zhu, Jian-Hua; Hu, Shen-Jiang

    2010-09-01

    Gap junctions, which serve as intercellular channels that allow the passage of ions and other small molecules between neighboring cells, play an important role in vital functions, including the regulation of cell growth, differentiation, and development. Statins, the 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitors, have been shown to inhibit the migration and proliferation of smooth muscle cells (SMCs) leading to an antiproliferative effect. Recent studies have shown that statins can reduce gap junction protein connexin43 (Cx43) expression both in vivo and in vitro. However, little work has been done on the effects of statins on gap junctional intercellular communication (GJIC). We hypothesized in this study that lovastatin inhibits vascular smooth muscle cells (VSMCs) migration through the inhibition of the GJIC. Rat aortic SMCs (RASMCs) were exposed to lovastatin. Vascular smooth muscle cells migration was then assessed with a Transwell migration assay. Gap junctional intercellular communication was determined by using fluorescence recovery after photobleaching (FRAP) analysis, which was performed with a laser-scanning confocal microscope. The migration of the cultured RASMCs were detected by Transwell system. Cell migration was dose-dependently inhibited with lovastatin. Compared with that in the control (110 ± 26), the number of migrated SMCs was significantly reduced to 72 ± 24 (P < .05), 62 ± 18 (P < .01), and 58 ± 19 (P < .01) at the concentration of 0.4, 2, and 10 umol/L, per field. The rate of fluorescence recovery (R) at 5 minutes after photobleaching was adopted as the functional index of GJIC. The R- value of cells exposed to lovastatin 10 umol/L for 48 hours was 24.38% ± 4.84%, whereas the cells in the control group had an R- value of 36.11% ± 10.53%, demonstrating that the GJIC of RASMCs was significantly inhibited by lovastatin (P < .01). Smaller concentrations of lovastatin 0.08 umol/L did not change gap junction coupling

  9. Chronic hemodynamic overload of the atria is an important factor for gap junction remodeling in human and rat hearts.

    PubMed

    Rucker-Martin, Catherine; Milliez, Paul; Tan, Sisareuth; Decrouy, Xavier; Recouvreur, Michel; Vranckx, Roger; Delcayre, Claude; Renaud, Jean-François; Dunia, Irene; Segretain, Dominique; Hatem, Stéphane N

    2006-10-01

    The expression and distribution of connexins is abnormal in a number of cardiac diseases, including atrial fibrillation, and is believed to favor conduction slowing and arrhythmia. Here, we studied the role of atrial structural remodeling in the disorganization of gap junctions and whether redistributed connexins can form new functional junction channels. Expression of connexin-43 (Cx43) was characterized by immunoblotting and immunohistochemistry in human right atrial specimens and in rat atria after myocardial infarction (MI). Gap junctions were studied by electron and 3-D microscopy, and myocyte-myocyte coupling was determined by Lucifer yellow dye transfer. In both chronically hemodynamically overloaded human atria in sinus rhythm and in dilated atria from MI-rats, Cx43 were dephosphorylated and redistributed from the intercalated disc to the lateral cell membranes as observed during atrial fibrillation. In MI-rats, the gap junctions at the intercalated disc were smaller (20% decrease) and contained very little Cx43 (0 or 1 gold particle vs. 42 to 98 in sham-operated rats). In the lateral membranes of myocytes, numerous connexon aggregates comprising non-phosphorylated Cx43 were observed. These connexon aggregates were in no case assembled into gap junction plaque-like structures. However, N-cadherin was well organized in the intercalated disc. There was very little myocyte-myocyte coupling in MI-rat atria and no myocyte-fibroblast coupling. Regression of the atrial remodeling was associated with the normalization of Cx43 localization. Structural alteration of the atrial myocardium is an important factor in the disorganization of connexins and gap junction. Moreover, redistributed Cx43 do not form junction channels.

  10. Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury.

    PubMed

    Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

    2010-08-01

    Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor alpha-glycyrrhetinic acid (alpha-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of alpha-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by alpha-GA. Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury.

  11. UNC-1 regulates gap junctions important to locomotion in C. elegans.

    PubMed

    Chen, Bojun; Liu, Qiang; Ge, Qian; Xie, Jia; Wang, Zhao-Wen

    2007-08-07

    In C. elegans, loss-of-function (lf) mutations of the stomatin-like protein (SLP) UNC-1 and the innexin UNC-9 inhibit locomotion [1, 2] and modulate sensitivity to volatile anesthetics [3, 4]. It was unknown why unc-1(lf) and unc-9(lf) mutants have similar phenotypes. We tested the hypothesis that UNC-1 is a regulator of gap junctions formed by UNC-9. Analyses of junctional currents between body-wall muscle cells showed that electrical coupling was inhibited to a similar degree in unc-1(lf), unc-9(lf), and unc-1(lf);unc-9(lf) double mutants, suggesting that UNC-1 and UNC-9 function together. Expression of Punc-1::DsRED2 and Punc-9::GFP transcriptional fusions suggests that unc-1 and unc-9 are coexpressed in neurons and body-wall muscle cells. Immunohistochemistry showed that UNC-1 and UNC-9 colocalized at intercellular junctions and that unc-1(lf) did not alter UNC-9 expression or subcellular localization. Bimolecular fluorescence complementation (BiFC) assays suggest that UNC-1 and UNC-9 are physically very close at intercellular junctions. Targeted rescue experiments suggest that UNC-9 and UNC-1 function predominantly in neurons to control locomotion. Thus, in addition to the recently reported function of regulating mechanosensitive ion channels [5, 6], SLPs might have a novel function of regulating gap junctions.

  12. Specific Cx43 phosphorylation events regulate gap junction turnover in vivo

    PubMed Central

    Solan, Joell L.; Lampe, Paul D.

    2014-01-01

    Gap junctions, composed of proteins from the connexin gene family, are highly dynamic structures that are regulated by kinase-mediated signaling pathways and interactions with other proteins. Phosphorylation of Connexin43 (Cx43) at different sites controls gap junction assembly, gap junction size and gap junction turnover. Here we present a model describing how Akt, mitogen activated protein kinase (MAPK) and src kinase coordinate to regulate rapid turnover of gap junctions. Specifically, Akt phosphorylates Cx43 at S373 eliminating interaction with zona occludens-1 (ZO-1) allowing gap junctions to enlarge. Then MAPK and src phosphorylate Cx43 to initiate turnover. We integrate published data with new data to test and refine this model. Finally, we propose that differential coordination of kinase activation and Cx43 phosphorylation controls the specific routes of disassembly, e.g., annular junction formation or gap junctions can potentially “unzip” and be internalized/endocytosed into the cell that produced each connexin. PMID:24508467

  13. Disruption of gap junctions attenuates aminoglycoside-elicited renal tubular cell injury

    PubMed Central

    Yao, Jian; Huang, Tao; Fang, Xin; Chi, Yuan; Zhu, Ying; Wan, Yigang; Matsue, Hiroyuki; Kitamura, Masanori

    2010-01-01

    BACKGROUND AND PURPOSE Gap junctions play important roles in the regulation of cell phenotype and in determining cell survival after various insults. Here, we investigated the role of gap junctions in aminoglycoside-induced injury to renal tubular cells. EXPERIMENTAL APPROACH Two tubular epithelial cell lines NRK-E52 and LLC-PK1 were compared for gap junction protein expression and function by immunofluorescent staining, Western blot and dye transfer assay. Cell viability after exposure to aminoglycosides was evaluated by WST assay. Gap junctions were modulated by transfection of the gap junction protein, connexin 43 (Cx43), use of Cx43 siRNA and gap junction inhibitors. KEY RESULTS NRK-E52 cells expressed abundant Cx43 and were functionally coupled by gap junctional intercellular communication (GJIC). Exposure of NRK-E52 cells to aminoglycosides, G418 and hygromycin, increased Cx43 phosphorylation and GJIC. The aminoglycosides also decreased cell viability that was prevented by gap junction inhibitors and Cx43 siRNA. LLC-PK1 cells were gap junction-deficient and resistant to aminoglycoside-induced cytotoxicity. Over-expression of a wild-type Cx43 converted LLC-PK1 cells to a drug-sensitive phenotype. The gap junction inhibitor α-glycyrrhetinic acid (α-GA) activated Akt in NRK-E52 cells. Inhibition of the Akt pathway enhanced cell toxicity to G418 and abolished the protective effects of α-GA. In addition, gentamycin-elicited cytotoxicity in NRK-E52 cells was also significantly attenuated by α-GA. CONCLUSION AND IMPLICATIONS Gap junctions contributed to the cytotoxic effects of aminoglycosides. Modulation of gap junctions could be a promising approach for prevention and treatment of aminoglycoside-induced renal tubular cell injury. PMID:20649601

  14. Design and characterization of the first peptidomimetic molecule that prevents acidification-induced closure of cardiac gap junctions

    PubMed Central

    Verma, Vandana; Larsen, Bjarne Due; Coombs, Wanda; Lin, Xianming; Sarrou, Eliana; Taffet, Steven M.; Delmar, Mario

    2010-01-01

    Background Gap junctions are potential targets for pharmacological intervention. We have previously developed a series of peptide sequences that prevent closure of Cx43 channels, bind to cardiac Cx43 and prevent acidification-induced uncoupling of cardiac gap junctions. Objective We aimed to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels. Methods Experiments were based on a combination of biochemical, spectroscopic and electrophysiological techniques, as well as molecular modeling of active pharmacophores with Cx43 activity. Results Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels, and pH gating of cardiac gap junctions. Conclusion Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacological agents for the treatment of a selected subset of cardiac arrhythmias. PMID:20601149

  15. Degradation of connexins and gap junctions

    PubMed Central

    Falk, Matthias M.; Kells, Rachael M.; Berthoud, Viviana M.

    2014-01-01

    Connexin proteins are short-lived within the cell, whether present in the secretory pathway or in gap junction plaques. Their levels can be modulated by their rate of degradation. Connexins, at different stages of assembly, are degraded through the proteasomal, endo-/lysosomal, and phago-/lysosomal pathways. In this review, we summarize the current knowledge about connexin and gap junction degradation including the signals and protein-protein interactions that participate in their targeting for degradation. PMID:24486527

  16. TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication

    PubMed Central

    Li, Hanjun; Spagnol, Gaelle; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

    2014-01-01

    ABSTRACT Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health. PMID:24849651

  17. The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells*

    PubMed Central

    Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J.; Wahl, James K.; Johnson, Keith R.; Mehta, Parmender P.

    2015-01-01

    Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. PMID:25548281

  18. The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

    PubMed

    Katoch, Parul; Mitra, Shalini; Ray, Anuttoma; Kelsey, Linda; Roberts, Brett J; Wahl, James K; Johnson, Keith R; Mehta, Parmender P

    2015-02-20

    Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The extracellular matrix component laminin promotes gap junction formation in the rat anterior pituitary gland.

    PubMed

    Horiguchi, Kotaro; Kouki, Tom; Fujiwara, Ken; Kikuchi, Motoshi; Yashiro, Takashi

    2011-03-01

    Folliculo-stellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. FS cells connect to each other not only by mechanical means, but also by gap junctional cell-to-cell communication. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture markedly change their shape, and form numerous interconnections with neighboring FS cells in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. Morphological and functional changes in cells are believed to be partly modified by matricrine signaling, by which ECM components function as cellular signals. In the present study, we examined whether gap junction formation between FS cells is affected by matricrine cues. A cell sorter was used to isolate FS cells from male S100b-GFP rat anterior pituitary for primary culture. We observed that mRNA and protein levels of connexin 43 in gap junction channels were clearly higher in the presence of laminin. In addition, we confirmed the formation of gap junctions between FS cells in primary culture by electron microscopy. Interestingly, we also observed that FS cells in the presence of laminin displayed well-developed rough endoplasmic reticulum and Golgi apparatus. Our findings suggest that, in anterior pituitary gland, FS cells may facilitate functional roles such as gap junctional cell-to-cell communication by matricrine signaling.

  20. Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness.

    PubMed

    Dalamon, Viviana; Fiori, Mariana C; Figueroa, Vania A; Oliva, Carolina A; Del Rio, Rodrigo; Gonzalez, Wendy; Canan, Jonathan; Elgoyhen, Ana B; Altenberg, Guillermo A; Retamal, Mauricio A

    2016-05-01

    Gap-junction channels (GJCs) are formed by head-to-head association of two hemichannels (HCs, connexin hexamers). HCs and GJCs are permeable to ions and hydrophilic molecules of up to Mr ~1 kDa. Hearing impairment of genetic origin is common, and mutations of connexin 26 (Cx26) are its major cause. We recently identified two novel Cx26 mutations in hearing-impaired subjects, L10P and G109V. L10P forms functional GJCs with slightly altered voltage dependence and HCs with decrease ATP/cationic dye selectivity. G109V does not form functional GJCs, but forms functional HCs with enhanced extracellular Ca(2+) sensitivity and subtle alterations in voltage dependence and ATP/cationic dye selectivity. Deafness associated with G109V could result from decreased GJCs activity, whereas deafness associated to L10P may have a more complex mechanism that involves changes in HC permeability.

  1. Distal gap junctions and active dendrites can tune network dynamics.

    PubMed

    Saraga, Fernanda; Ng, Leo; Skinner, Frances K

    2006-03-01

    Gap junctions allow direct electrical communication between CNS neurons. From theoretical and modeling studies, it is well known that although gap junctions can act to synchronize network output, they can also give rise to many other dynamic patterns including antiphase and other phase-locked states. The particular network pattern that arises depends on cellular, intrinsic properties that affect firing frequencies as well as the strength and location of the gap junctions. Interneurons or GABAergic neurons in hippocampus are diverse in their cellular characteristics and have been shown to have active dendrites. Furthermore, parvalbumin-positive GABAergic neurons, also known as basket cells, can contact one another via gap junctions on their distal dendrites. Using two-cell network models, we explore how distal electrical connections affect network output. We build multi-compartment models of hippocampal basket cells using NEURON and endow them with varying amounts of active dendrites. Two-cell networks of these model cells as well as reduced versions are explored. The relationship between intrinsic frequency and the level of active dendrites allows us to define three regions based on what sort of network dynamics occur with distal gap junction coupling. Weak coupling theory is used to predict the delineation of these regions as well as examination of phase response curves and distal dendritic polarization levels. We find that a nonmonotonic dependence of network dynamic characteristics (phase lags) on gap junction conductance occurs. This suggests that distal electrical coupling and active dendrite levels can control how sensitive network dynamics are to gap junction modulation. With the extended geometry, gap junctions located at more distal locations must have larger conductances for pure synchrony to occur. Furthermore, based on simulations with heterogeneous networks, it may be that one requires active dendrites if phase-locking is to occur in networks formed

  2. Physiological Role of Gap-Junctional Hemichannels

    PubMed Central

    Quist, Arjan Pieter; Rhee, Seung Keun; Lin, Hai; Lal, Ratneshwar

    2000-01-01

    Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation. PMID:10704454

  3. Models and methods for in vitro testing of hepatic gap junctional communication.

    PubMed

    Maes, Michaël; Yanguas, Sara Crespo; Willebrords, Joost; Vinken, Mathieu

    2015-12-25

    Inherent to their pivotal roles in controlling all aspects of the liver cell life cycle, hepatocellular gap junctions are frequently disrupted upon impairment of the homeostatic balance, as occurs during liver toxicity. Hepatic gap junctions, which are mainly built up by connexin32, are specifically targeted by tumor promoters and epigenetic carcinogens. This renders inhibition of gap junction functionality a suitable indicator for the in vitro detection of nongenotoxic hepatocarcinogenicity. The establishment of a reliable liver gap junction inhibition assay for routine in vitro testing purposes requires a cellular system in which gap junctions are expressed at an in vivo-like level as well as an appropriate technique to probe gap junction activity. Both these models and methods are discussed in the current paper, thereby focusing on connexin32-based gap junctions. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells

    PubMed Central

    Cheng, Catherine; Nowak, Roberta B.; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K.; Lo, Woo-Kuen; Mathias, Richard T.

    2015-01-01

    The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1−/−;CP49−/− double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. PMID:25740157

  5. Lens ion homeostasis relies on the assembly and/or stability of large connexin 46 gap junction plaques on the broad sides of differentiating fiber cells.

    PubMed

    Cheng, Catherine; Nowak, Roberta B; Gao, Junyuan; Sun, Xiurong; Biswas, Sondip K; Lo, Woo-Kuen; Mathias, Richard T; Fowler, Velia M

    2015-05-15

    The eye lens consists of layers of tightly packed fiber cells, forming a transparent and avascular organ that is important for focusing light onto the retina. A microcirculation system, facilitated by a network of gap junction channels composed of connexins 46 and 50 (Cx46 and Cx50), is hypothesized to maintain and nourish lens fiber cells. We measured lens impedance in mice lacking tropomodulin 1 (Tmod1, an actin pointed-end capping protein), CP49 (a lens-specific intermediate filament protein), or both Tmod1 and CP49. We were surprised to find that simultaneous loss of Tmod1 and CP49, which disrupts cytoskeletal networks in lens fiber cells, results in increased gap junction coupling resistance, hydrostatic pressure, and sodium concentration. Protein levels of Cx46 and Cx50 in Tmod1(-/-);CP49(-/-) double-knockout (DKO) lenses were unchanged, and electron microscopy revealed normal gap junctions. However, immunostaining and quantitative analysis of three-dimensional confocal images showed that Cx46 gap junction plaques are smaller and more dispersed in DKO differentiating fiber cells. The localization and sizes of Cx50 gap junction plaques in DKO fibers were unaffected, suggesting that Cx46 and Cx50 form homomeric channels. We also demonstrate that gap junction plaques rest in lacunae of the membrane-associated actin-spectrin network, suggesting that disruption of the actin-spectrin network in DKO fibers may interfere with gap junction plaque accretion into micrometer-sized domains or alter the stability of large plaques. This is the first work to reveal that normal gap junction plaque localization and size are associated with normal lens coupling conductance. Copyright © 2015 the American Physiological Society.

  6. Gap junction connexins in female reproductive organs: implications for women's reproductive health.

    PubMed

    Winterhager, Elke; Kidder, Gerald M

    2015-01-01

    Connexins comprise a family of ~20 proteins that form intercellular membrane channels (gap junction channels) providing a direct route for metabolites and signalling molecules to pass between cells. This review provides a critical analysis of the evidence for essential roles of individual connexins in female reproductive function, highlighting implications for women's reproductive health. No systematic review has been carried out. Published literature from the past 35 years was surveyed for research related to connexin involvement in development and function of the female reproductive system. Because of the demonstrated utility of genetic manipulation for elucidating connexin functions in various organs, much of the cited information comes from research with genetically modified mice. In some cases, a distinction is drawn between connexin functions clearly related to the formation of gap junction channels and those possibly linked to non-channel roles. Based on work with mice, several connexins are known to be required for female reproductive functions. Loss of connexin43 (CX43) causes an oocyte deficiency, and follicles lacking or expressing less CX43 in granulosa cells exhibit reduced growth, impairing fertility. CX43 is also expressed in human cumulus cells and, in the context of IVF, has been correlated with pregnancy outcome, suggesting that this connexin may be a determinant of oocyte and embryo quality in women. Loss of CX37, which exclusively connects oocytes with granulosa cells in the mouse, caused oocytes to cease growing without acquiring meiotic competence. Blocking of CX26 channels in the uterine epithelium disrupted implantation whereas loss or reduction of CX43 expression in the uterine stroma impaired decidualization and vascularization in mouse and human. Several connexins are important in placentation and, in the human, CX43 is a key regulator of the fusogenic pathway from the cytotrophoblast to the syncytiotrophoblast, ensuring placental growth

  7. Gap junction plasticity as a mechanism to regulate network-wide oscillations

    PubMed Central

    Nicola, Wilten; Clopath, Claudia

    2018-01-01

    Cortical oscillations are thought to be involved in many cognitive functions and processes. Several mechanisms have been proposed to regulate oscillations. One prominent but understudied mechanism is gap junction coupling. Gap junctions are ubiquitous in cortex between GABAergic interneurons. Moreover, recent experiments indicate their strength can be modified in an activity-dependent manner, similar to chemical synapses. We hypothesized that activity-dependent gap junction plasticity acts as a mechanism to regulate oscillations in the cortex. We developed a computational model of gap junction plasticity in a recurrent cortical network based on recent experimental findings. We showed that gap junction plasticity can serve as a homeostatic mechanism for oscillations by maintaining a tight balance between two network states: asynchronous irregular activity and synchronized oscillations. This homeostatic mechanism allows for robust communication between neuronal assemblies through two different mechanisms: transient oscillations and frequency modulation. This implies a direct functional role for gap junction plasticity in information transmission in cortex. PMID:29529034

  8. Interferon-gamma inhibits intestinal restitution by preventing gap junction communication between enterocytes.

    PubMed

    Leaphart, Cynthia L; Qureshi, Faisal; Cetin, Selma; Li, Jun; Dubowski, Theresa; Baty, Catherine; Batey, Catherine; Beer-Stolz, Donna; Guo, Fengli; Murray, Sandra A; Hackam, David J

    2007-06-01

    Necrotizing enterocolitis (NEC) is characterized by interferon-gamma (IFN-gamma) release and inadequate intestinal restitution. Because enterocytes migrate together, mucosal healing may require interenterocyte communication via connexin 43-mediated gap junctions. We hypothesize that enterocyte migration requires interenterocyte communication, that IFN impairs migration by impairing connexin 43, and that impaired healing during NEC is associated with reduced gap junctions. NEC was induced in Swiss-Webster or IFN(-/-) mice, and restitution was determined in the presence of the gap junction inhibitor oleamide, or via time-lapse microscopy of IEC-6 cells. Connexin 43 expression, trafficking, and localization were detected in cultured or primary enterocytes or mouse or human intestine by confocal microscopy and (35)S-labeling, and gap junction communication was assessed using live microscopy with oleamide or connexin 43 siRNA. Enterocytes expressed connexin 43 in vitro and in vivo, and exchanged fluorescent dye via gap junctions. Gap junction inhibition significantly reduced enterocyte migration in vitro and in vivo. NEC was associated with IFN release and loss of enterocyte connexin 43 expression. IFN inhibited enterocyte migration by reducing gap junction communication through the dephosphorylation and internalization of connexin 43. Gap junction inhibition significantly increased NEC severity, whereas reversal of the inhibitory effects of IFN on gap junction communication restored enterocyte migration after IFN exposure. Strikingly, IFN(-/-) mice were protected from the development of NEC, and showed restored connexin 43 expression and intestinal restitution. IFN inhibits enterocyte migration by preventing interenterocyte gap junction communication. Connexin 43 loss may provide insights into the development of NEC, in which restitution is impaired.

  9. TEMPORAL CHANGE IN GAP JUNCTION FUNCTION IN PRIMARY HEPATOCYTES

    EPA Science Inventory

    TEMPORAL CHANGES IN GAP JUNCTION FUNCTION IN PRIMARY *

    The objective of this study was to examine the reduction in gap junction communication (GJC) in primary hepatocytes due to coincident melatonin and magnetic field treatments to determine if these conditions could prov...

  10. Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells.

    PubMed

    Nagasawa, Kunihiko; Chiba, Hideki; Fujita, Hiroki; Kojima, Takashi; Saito, Tsuyoshi; Endo, Toshiaki; Sawada, Norimasa

    2006-07-01

    Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed. Copyright 2006 Wiley-Liss, Inc.

  11. Application of stochastic automata networks for creation of continuous time Markov chain models of voltage gating of gap junction channels.

    PubMed

    Snipas, Mindaugas; Pranevicius, Henrikas; Pranevicius, Mindaugas; Pranevicius, Osvaldas; Paulauskas, Nerijus; Bukauskas, Feliksas F

    2015-01-01

    The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ~20 times.

  12. Application of Stochastic Automata Networks for Creation of Continuous Time Markov Chain Models of Voltage Gating of Gap Junction Channels

    PubMed Central

    Pranevicius, Henrikas; Pranevicius, Mindaugas; Pranevicius, Osvaldas; Bukauskas, Feliksas F.

    2015-01-01

    The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ∼20 times. PMID:25705700

  13. The E3 ubiquitin ligase NEDD4 induces endocytosis and lysosomal sorting of connexin 43 to promote loss of gap junctions.

    PubMed

    Totland, Max Z; Bergsland, Christian H; Fykerud, Tone A; Knudsen, Lars M; Rasmussen, Nikoline L; Eide, Peter W; Yohannes, Zeremariam; Sørensen, Vigdis; Brech, Andreas; Lothe, Ragnhild A; Leithe, Edward

    2017-09-01

    Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. © 2017. Published by The Company of Biologists Ltd.

  14. Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis.

    PubMed

    Kikuchi, T; Adams, J C; Paul, D L; Kimura, R S

    1994-09-01

    The distribution of gap junctions within the vestibular labyrinth was investigated using immunohistochemistry and transmission electron microscopy. Connexin26-like immunoreactivity was observed among supporting cells in each vestibular sensory epithelium. Reaction product was also present in the transitional epithelium of each vestibular endorgan and in the planum semilunatum of crista ampullaris. No connexin26-like immunoreactivity was observed among thin wall epithelial cells or among vestibular dark cells. In addition, fibrocytes within vestibular connective tissue were positively immunostained. Reaction product was also detected in the melanocyte area just beneath dark cells. Ultrastructural observations indicated that a gap junction network of vestibular supporting cells extends to the transitional epithelium and planum semilunatum and forms an isolated epithelial cell gap junction system in each vestibular endorgan. In contrast, no gap junctions were found among wall epithelial cells or among dark cells. Fibrocytes and melanocytes were coupled by gap junctions and belong to the connective tissue cell gap junction system, which is continuous throughout the vestibular system and the cochlea. The possible functional significance of these gap junction systems is discussed.

  15. Phylogenetic and bioinformatic analysis of gap junction-related proteins, innexins, pannexins and connexins.

    PubMed

    Fushiki, Daisuke; Hamada, Yasuo; Yoshimura, Ryoichi; Endo, Yasuhisa

    2010-04-01

    All multi-cellular animals, including hydra, insects and vertebrates, develop gap junctions, which communicate directly with neighboring cells. Gap junctions consist of protein families called connexins in vertebrates and innexins in invertebrates. Connexins and innexins have no homology in their amino acid sequence, but both are thought to have some similar characteristics, such as a tetra-membrane-spanning structure, formation of a channel by hexamer, and transmission of small molecules (e.g. ions) to neighboring cells. Pannexins were recently identified as a homolog of innexins in vertebrate genomes. Although pannexins are thought to share the function of intercellular communication with connexins and innexins, there is little information about the relationship among these three protein families of gap junctions. We phylgenetically and bioinformatically examined these protein families and other tetra-membrane-spanning proteins using a database and three analytical softwares. The clades formed by pannexin families do not belong to the species classification but do to paralogs of each member of pannexins. Amino acid sequences of pannexins are closely related to those of innexins but less to those of connexins. These data suggest that innexins and pannexins have a common origin, but the relationship between innexins/pannexins and connexins is as slight as that of other tetra-membrane-spanning members.

  16. Fixed-Gap Tunnel Junction for Reading DNA Nucleotides

    PubMed Central

    2015-01-01

    Previous measurements of the electronic conductance of DNA nucleotides or amino acids have used tunnel junctions in which the gap is mechanically adjusted, such as scanning tunneling microscopes or mechanically controllable break junctions. Fixed-junction devices have, at best, detected the passage of whole DNA molecules without yielding chemical information. Here, we report on a layered tunnel junction in which the tunnel gap is defined by a dielectric layer, deposited by atomic layer deposition. Reactive ion etching is used to drill a hole through the layers so that the tunnel junction can be exposed to molecules in solution. When the metal electrodes are functionalized with recognition molecules that capture DNA nucleotides via hydrogen bonds, the identities of the individual nucleotides are revealed by characteristic features of the fluctuating tunnel current associated with single-molecule binding events. PMID:25380505

  17. Spinal astrocyte gap junctions contribute to oxaliplatin-induced mechanical hypersensitivity.

    PubMed

    Yoon, Seo-Yeon; Robinson, Caleb R; Zhang, Haijun; Dougherty, Patrick M

    2013-02-01

    Spinal glial cells contribute to the development of many types of inflammatory and neuropathic pain. Here the contribution of spinal astrocytes and astrocyte gap junctions to oxaliplatin-induced mechanical hypersensitivity was explored. The expression of glial fibrillary acidic protein (GFAP) in spinal dorsal horn was significantly increased at day 7 but recovered at day 14 after oxaliplatin treatment, suggesting a transient activation of spinal astrocytes by chemotherapy. Astrocyte-specific gap junction protein connexin 43 (Cx43) was significantly increased in dorsal horn at both day 7 and day 14 following chemotherapy, but neuronal (connexin 36 [Cx36]) and oligodendrocyte (connexin 32 [Cx32]) gap junction proteins did not show any change. Blockade of astrocyte gap junction with carbenoxolone (CBX) prevented oxaliplatin-induced mechanical hypersensitivity in a dose-dependent manner and the increase of spinal GFAP expression, but had no effect once the mechanical hypersensitivity induced by oxaliplatin had fully developed. These results suggest that oxaliplatin chemotherapy induces the activation of spinal astrocytes and this is accompanied by increased expression of astrocyte-astrocyte gap junction connections via Cx43. These alterations in spinal astrocytes appear to contribute to the induction but not the maintenance of oxaliplatin-induced mechanical hypersensitivity. Combined, these results suggest that targeting spinal astrocyte/astrocyte-specific gap junction could be a new therapeutic strategy to prevent oxaliplatin-induced neuropathy. Spinal astrocytes but not microglia were recently shown to be recruited in paclitaxel-related chemoneuropathy. Here, spinal astrocyte gap junctions are shown to play an important role in the induction of oxaliplatin neuropathy. Copyright © 2013 American Pain Society. Published by Elsevier Inc. All rights reserved.

  18. Bar dynamics and channel junctions in scale-experiments of estuaries

    NASA Astrophysics Data System (ADS)

    Leuven, J.; Braat, L.; van Dijk, W. M.; Haas, T. D.; Kleinhans, M. G.

    2017-12-01

    The evolution of channels and bars in estuaries has high socio-economic relevance, with strong implications for navigation, dredging and ecology. However, the spatial and temporal evolution of channels and bars in estuaries is poorly understood. Here, we study feedbacks of bar morphodynamics on widening and narrowing of estuaries. Therefore, we conducted an experiment in a 20 m long and 3 m wide tilting flume (the 'Metronome'), in which we monitored the evolution of a self-formed estuary that developed from an intial straight channel into an irregular planform with multiple channels, braided bars and a meandering ebb channel. At locations where the estuary width is confined, major channel junctions occur, while the zones between the junctions are characterised by high braiding indices, periodically migrating channels and a relatively large estuary width. The junction locations were forced by the in- and outflow locations on the sides of the ebb-tidal delta and at the location where the channel pattern transitions from multiple channels into a single channel. In the middle of the estuary, self-confinement occurred by sedimentation on the sides of the estuary, which caused another major junction. The channel orientation at the junctions steers the morphodynamics of channels and bars immediately landward and seaward, because the orientation of inflow from the ebb-tidal delta and landward river perpetually varies. In natural systems major junction locations are mostly forced by inherited geology or human engineering. However, this study concludes that even without external forcing, the estuary planform will not converge to an ideal shape but will self-confine at major junctions and widens in the adjacent zones, resulting in an irregular planform shape.

  19. CHLORAL HYDRATE DECREASES GAP JUNCTION COMMUNICATION IN RAT LIVER EPITHELIAL CELLS

    EPA Science Inventory

    Chloral hydrate decreases gap junction communication in rat liver epithelial cells

    Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Connexins (Cx) that make up these junctions are composed of a closely related group of m...

  20. Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

    PubMed

    Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

    2012-01-20

    The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

  1. Gap Junctions and Cancer: Communicating for 50 Years’

    PubMed Central

    Aasen, Trond; Mesnil, Marc; Naus, Christian C.; Lampe, Paul D.; Laird, Dale W.

    2017-01-01

    Fifty years ago, tumour cells were found to lack electrical coupling, leading to the hypothesis that loss of direct intercellular communication is commonly associated with cancer onset and progression. Subsequent studies linked this phenomenon to gap junctions composed of connexin proteins. While many studies support the notion that connexins are tumour suppressors, recent evidence suggests that, in some tumour types, they may facilitate specific stages of tumour progression through both junctional and non-junctional signalling pathways. This Timeline article highlights the milestones connecting gap junctions to cancer, and underscores important unanswered questions, controversies and therapeutic opportunities in the field. PMID:27782134

  2. Robustness effect of gap junctions between Golgi cells on cerebellar cortex oscillations

    PubMed Central

    2011-01-01

    Background Previous one-dimensional network modeling of the cerebellar granular layer has been successfully linked with a range of cerebellar cortex oscillations observed in vivo. However, the recent discovery of gap junctions between Golgi cells (GoCs), which may cause oscillations by themselves, has raised the question of how gap-junction coupling affects GoC and granular-layer oscillations. To investigate this question, we developed a novel two-dimensional computational model of the GoC-granule cell (GC) circuit with and without gap junctions between GoCs. Results Isolated GoCs coupled by gap junctions had a strong tendency to generate spontaneous oscillations without affecting their mean firing frequencies in response to distributed mossy fiber input. Conversely, when GoCs were synaptically connected in the granular layer, gap junctions increased the power of the oscillations, but the oscillations were primarily driven by the synaptic feedback loop between GoCs and GCs, and the gap junctions did not change oscillation frequency or the mean firing rate of either GoCs or GCs. Conclusion Our modeling results suggest that gap junctions between GoCs increase the robustness of cerebellar cortex oscillations that are primarily driven by the feedback loop between GoCs and GCs. The robustness effect of gap junctions on synaptically driven oscillations observed in our model may be a general mechanism, also present in other regions of the brain. PMID:22330240

  3. Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

    PubMed Central

    1995-01-01

    Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

  4. Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

    PubMed

    Veeraraghavan, Rengasayee; Lin, Joyce; Hoeker, Gregory S; Keener, James P; Gourdie, Robert G; Poelzing, Steven

    2015-10-01

    It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated "ephaptic" mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 μM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 μM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.

  5. MicroRNA Intercellular Transfer and Bioelectrical Regulation of Model Multicellular Ensembles by the Gap Junction Connectivity.

    PubMed

    Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

    2017-08-17

    We have studied theoretically the microRNA (miRNA) intercellular transfer through voltage-gated gap junctions in terms of a biophysically grounded system of coupled differential equations. Instead of modeling a specific system, we use a general approach describing the interplay between the genetic mechanisms and the single-cell electric potentials. The dynamics of the multicellular ensemble are simulated under different conditions including spatially inhomogeneous transcription rates and local intercellular transfer of miRNAs. These processes result in spatiotemporal changes of miRNA, mRNA, and ion channel protein concentrations that eventually modify the bioelectrical states of small multicellular domains because of the ensemble average nature of the electrical potential. The simulations allow a qualitative understanding of the context-dependent nature of the effects observed when specific signaling molecules are transferred through gap junctions. The results suggest that an efficient miRNA intercellular transfer could permit the spatiotemporal control of small cellular domains by the conversion of single-cell genetic and bioelectric states into multicellular states regulated by the gap junction interconnectivity.

  6. Methamphetamine compromises gap junctional communication in astrocytes and neurons.

    PubMed

    Castellano, Paul; Nwagbo, Chisom; Martinez, Luis R; Eugenin, Eliseo A

    2016-05-01

    Methamphetamine (meth) is a central nervous system (CNS) stimulant that results in psychological and physical dependency. The long-term effects of meth within the CNS include neuronal plasticity changes, blood-brain barrier compromise, inflammation, electrical dysfunction, neuronal/glial toxicity, and an increased risk to infectious diseases including HIV. Most of the reported meth effects in the CNS are related to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters, especially dopamine, norepinephrine, and epinephrine. However, little is known about the effects of meth on connexin (Cx) containing channels, such as gap junctions (GJ) and hemichannels (HC). We examined the effects of meth on Cx expression, function, and its role in NeuroAIDS. We found that meth altered Cx expression and localization, decreased GJ communication between neurons and astrocytes, and induced the opening of Cx43/Cx36 HC. Furthermore, we found that these changes in GJ and HC induced by meth treatment were mediated by activation of dopamine receptors, suggesting that dysregulation of dopamine signaling induced by meth is essential for GJ and HC compromise. Meth-induced changes in GJ and HC contributed to amplified CNS toxicity by dysregulating glutamate metabolism and increasing the susceptibility of neurons and astrocytes to bystander apoptosis induced by HIV. Together, our results indicate that connexin containing channels, GJ and HC, are essential in the pathogenesis of meth and increase the sensitivity of the CNS to HIV CNS disease. Methamphetamine (meth) is an extremely addictive central nervous system stimulant. Meth reduced gap junctional (GJ) communication by inducing internalization of connexin-43 (Cx43) in astrocytes and reducing expression of Cx36 in neurons by a mechanism involving activation of dopamine receptors (see cartoon). Meth-induced changes in Cx containing channels increased extracellular levels of glutamate and resulted in higher

  7. Heterotypic gap junctions at glutamatergic mixed synapses are abundant in goldfish brain

    PubMed Central

    Rash, John E.; Kamasawa, Naomi; Vanderpool, Kimberly G.; Yasumura, Thomas; O'Brien, John; Nannapaneni, Srikant; Pereda, Alberto E.; Nagy, James I.

    2014-01-01

    Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at “large myelinated club ending” synapses on Mauthner cells of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical “pre-potentials” immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified “mixed” (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with Cx35 restricted to axon terminal hemiplaques and Cx34.7 restricted to apposing Mauthner cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of Cx36 on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or

  8. Three-dimensional modeling and quantitative analysis of gap junction distributions in cardiac tissue.

    PubMed

    Lackey, Daniel P; Carruth, Eric D; Lasher, Richard A; Boenisch, Jan; Sachse, Frank B; Hitchcock, Robert W

    2011-11-01

    Gap junctions play a fundamental role in intercellular communication in cardiac tissue. Various types of heart disease including hypertrophy and ischemia are associated with alterations of the spatial arrangement of gap junctions. Previous studies applied two-dimensional optical and electron-microscopy to visualize gap junction arrangements. In normal cardiomyocytes, gap junctions were primarily found at cell ends, but can be found also in more central regions. In this study, we extended these approaches toward three-dimensional reconstruction of gap junction distributions based on high-resolution scanning confocal microscopy and image processing. We developed methods for quantitative characterization of gap junction distributions based on analysis of intensity profiles along the principal axes of myocytes. The analyses characterized gap junction polarization at cell ends and higher-order statistical image moments of intensity profiles. The methodology was tested in rat ventricular myocardium. Our analysis yielded novel quantitative data on gap junction distributions. In particular, the analysis demonstrated that the distributions exhibit significant variability with respect to polarization, skewness, and kurtosis. We suggest that this methodology provides a quantitative alternative to current approaches based on visual inspection, with applications in particular in characterization of engineered and diseased myocardium. Furthermore, we propose that these data provide improved input for computational modeling of cardiac conduction.

  9. Bioelectrical coupling in multicellular domains regulated by gap junctions: A conceptual approach.

    PubMed

    Cervera, Javier; Pietak, Alexis; Levin, Michael; Mafe, Salvador

    2018-04-21

    We review the basic concepts involved in bioelectrically-coupled multicellular domains, focusing on the role of membrane potentials (V mem ). In the first model, single-cell V mem is modulated by two generic polarizing and depolarizing ion channels, while intercellular coupling is implemented via voltage-gated gap junctions. Biochemical and bioelectrical signals are integrated via a feedback loop between V mem and the transcription and translation of a protein forming an ion channel. The effective rate constants depend on the single-cell V mem because these potentials modulate the local concentrations of signaling molecules and ions. This electrochemically-based idealization of the complex biophysical problem suggests that the spatio-temporal map of single-cell potentials can influence downstream patterning processes by means of the voltage-gated gap junction interconnectivity, much as in the case of electronic devices where the control of electric potentials and currents allows the local modulation of the circuitry to achieve full functionality. An alternative theoretical approach, the BioElectrical Tissue Simulation Engine (BETSE), is also presented. The BETSE modeling environment utilizes finite volume techniques to simulate bioelectric states from the perspective of ion concentrations and fluxes. This model has been successfully applied to make predictions and explain experimental observations in a variety of embryonic, regenerative, and oncogenic contexts. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations

    PubMed Central

    Oviedo‐orta, E; Hoy, T; Evans, W H

    2000-01-01

    The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil‐derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil‐derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription–polymerase chain reaction (RT–PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and α‐glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co‐cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T‐ and B‐lymphocyte co‐cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes. PMID:10792506

  11. Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts.

    PubMed

    Sundset, Rune; Cooper, Marie; Mikalsen, Svein-Ole; Ytrehus, Kirsti

    2004-01-01

    Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.

  12. Gap Junctions and Connexin Hemichannels Underpin Haemostasis and Thrombosis

    PubMed Central

    Vaiyapuri, Sakthivel; Jones, Chris I.; Sasikumar, Parvathy; Moraes, Leonardo A.; Munger, Stephanie J.; Wright, Joy R.; Ali, Marfoua S.; Sage, Tanya; Kaiser, William J.; Tucker, Katherine L.; Stain, Christopher J.; Bye, Alexander P.; Jones, Sarah; Oviedo-Orta, Ernesto; Simon, Alexander M.; Mahaut-Smith, Martyn P.; Gibbins, Jonathan M.

    2012-01-01

    Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets. PMID:22528526

  13. Interactions of Pannexin1 channels with purinergic and NMDA receptor channels.

    PubMed

    Li, Shuo; Bjelobaba, Ivana; Stojilkovic, Stanko S

    2018-01-01

    Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Published by Elsevier B.V.

  14. Gap Junctional Communication in Morphogenesis

    PubMed Central

    Levin, Michael

    2007-01-01

    Gap junctions permit the direct passage of small molecules from the cytosol of one cell to that of its neighbor, and thus form a system of cell-cell communication that exists alongside familiar secretion/receptor signaling. Because of the rich potential for regulation of junctional conductance, and directional and molecular gating (specificity), gap junctional communication (GJC) plays a crucial role in many aspects of normal tissue physiology. However, the most exciting role for GJC is in the regulation of information flow that takes place during embryonic development, regeneration, and tumor progression. The molecular mechanisms by which GJC establishes local and long-range instructive morphogenetic cues are just beginning to be understood. This review summarizes the current knowledge of the involvement of GJC in the patterning of both vertebrate and invertebrate systems and discusses in detail several morphogenetic systems in which the properties of this signaling have been molecularly characterized. One model consistent with existing data in the fields of vertebrate left-right patterning and anterior-posterior polarity in flatworm regeneration postulates electrophoretically-guided movement of small molecule morphogens through long-range GJC paths. The discovery of mechanisms controlling embryonic and regenerative GJC-mediated signaling, and identification of the downstream targets of GJC-permeable molecules, represent exciting next areas of research in this fascinating field. PMID:17481700

  15. Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans.

    PubMed

    Meng, Lingfeng; Zhang, Albert; Jin, Yishi; Yan, Dong

    2016-10-21

    Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4 . Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.

  16. Kinase programs spatiotemporally regulate gap junction assembly and disassembly: effects on wound repair

    PubMed Central

    Solan, Joell L.; Lampe, Paul D.

    2016-01-01

    Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43’s half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing. PMID:26706150

  17. Kinase programs spatiotemporally regulate gap junction assembly and disassembly: Effects on wound repair.

    PubMed

    Solan, Joell L; Lampe, Paul D

    2016-02-01

    Gap junctions are highly ordered plasma membrane domains that are constantly assembled, remodeled and turned over due to the short half-life of connexins, the integral membrane proteins that form gap junctions. Connexin 43 (Cx43), by far the most widely expressed connexin, is phosphorylated at multiple serine residues in the cytoplasmic, C-terminal region allowing for exquisite cellular control over gap junctional communication. This is evident during epidermal wounding where spatiotemporal changes in connexin expression occur as cells are instructed whether to die, proliferate or migrate to promote repair. Early gap junctional communication is required for initiation of keratinocyte migration, but accelerated Cx43 turnover is also critical for proper wound healing at later stages. These events are controlled via a "kinase program" where sequential phosphorylation of Cx43 leads to reductions in Cx43's half-life and significant depletion of gap junctions from the plasma membrane within several hours. The complex regulation of gap junction assembly and turnover affords several steps where intervention might speed wound healing. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Dependence of paranodal junctional gap width on transverse bands.

    PubMed

    Rosenbluth, Jack; Petzold, Chris; Peles, Elior

    2012-08-15

    Mouse mutants with paranodal junctional (PNJ) defects display variable degrees of neurological impairment. In this study we compare control paranodes with those from three mouse mutants that differ with respect to a conspicuous PNJ component, the transverse bands (TBs). We hypothesize that TBs link the apposed junctional membranes together at a fixed distance and thereby determine the width of the junctional gap, which may in turn determine the extent to which nodal action currents can be short-circuited underneath the myelin sheath. Electron micrographs of aldehyde-fixed control PNJs, in which TBs are abundant, show a consistent junctional gap of ∼3.5 nm. In Caspr-null PNJs, which lack TBs entirely, the gap is wider (∼6-7 nm) and more variable. In CST-null PNJs, which have only occasional TBs, the mean PNJ gap width is comparable to that in Caspr-null mice. In the shaking mutant, in contrast, which has approximately 60% of the normal complement of TBs, mean PNJ gap width is not significantly different from that in controls. Correspondingly, shaking mice are much less impaired neurologically than either Caspr-null or CST-null mice. We conclude that in the absence or gross diminution of TBs, mean PNJ gap width increases significantly and suggest that this difference could underlie some of the neurological impairment seen in those mutants. Surprisingly, even in the absence of TBs, paranodes are to some extent maintained in their usual form, implying that in addition to TBs, other factors govern the formation and maintenance of overall paranodal structure. Copyright © 2012 Wiley Periodicals, Inc.

  19. Relationship between connexin expression and gap-junction resistivity in human atrial myocardium.

    PubMed

    Dhillon, Paramdeep S; Chowdhury, Rasheda A; Patel, Pravina M; Jabr, Rita; Momin, Aziz U; Vecht, Joshua; Gray, Rosaire; Shipolini, Alex; Fry, Christopher H; Peters, Nicholas S

    2014-04-01

    The relative roles of the gap-junctional proteins connexin40 (Cx40) and connexin43 (Cx43) in determining human atrial myocardial resistivity is unknown. In addressing the hypothesis that changing relative expression of Cx40 and Cx43 underlies an increase in human atrial myocardial resistivity with age, this relationship was investigated by direct ex vivo measurement of gap-junctional resistivity and quantitative connexin immunoblotting and immunohistochemistry. Oil-gap impedance measurements were performed to determine resistivity of the intracellular pathway (Ri), which correlated with total Cx40 quantification by Western blotting (rs=0.64, P<0.01, n=20). Specific gap-junctional resistivity (Rj) correlated not only with Western immunoquantification of Cx40 (rs=0.63, P=0.01, n=20), but also more specifically, with the Cx40 fraction localized to the intercalated disks on immunohistochemical quantification (rs=0.66, P=0.02, n=12). Although Cx43 expression showed no correlation with resistivity values, the proportional expression of the 2 connexins, (Cx40/[Cx40+Cx43]) correlated with Ri and Rj (rs=0.58, P<0.01 for Ri and rs=0.51, P=0.02 for Rj). Advancing age was associated with a rise in Ri (rs=0.77, P<0.0001), Rj (rs=0.65, P<0.001, n=23), Cx40 quantity (rs=0.54, P=0.01, n=20), and Cx40 gap-junction protein per unit area of en face disk (rs=0.61, P=0.02, n=12). Cx40 is associated with human right atrial gap-junctional resistivity such that increased total, gap-junctional, and proportional Cx40 expression increases gap-junctional resistivity. Accordingly, advancing age is associated with an increase in Cx40 expression and a corresponding increase in gap-junctional resistivity. These findings are the first to demonstrate this relationship and a mechanistic explanation for changing atrial conduction and age-related arrhythmic tendency.

  20. Emergent central pattern generator behavior in gap-junction-coupled Hodgkin-Huxley style neuron model.

    PubMed

    Horn, Kyle G; Memelli, Heraldo; Solomon, Irene C

    2012-01-01

    Most models of central pattern generators (CPGs) involve two distinct nuclei mutually inhibiting one another via synapses. Here, we present a single-nucleus model of biologically realistic Hodgkin-Huxley neurons with random gap junction coupling. Despite no explicit division of neurons into two groups, we observe a spontaneous division of neurons into two distinct firing groups. In addition, we also demonstrate this phenomenon in a simplified version of the model, highlighting the importance of afterhyperpolarization currents (I(AHP)) to CPGs utilizing gap junction coupling. The properties of these CPGs also appear sensitive to gap junction conductance, probability of gap junction coupling between cells, topology of gap junction coupling, and, to a lesser extent, input current into our simulated nucleus.

  1. Gap junctional coupling in the vertebrate retina: variations on one theme?

    PubMed

    Völgyi, Béla; Kovács-Oller, Tamás; Atlasz, Tamás; Wilhelm, Márta; Gábriel, Róbert

    2013-05-01

    Gap junctions connect cells in the bodies of all multicellular organisms, forming either homologous or heterologous (i.e. established between identical or different cell types, respectively) cell-to-cell contacts by utilizing identical (homotypic) or different (heterotypic) connexin protein subunits. Gap junctions in the nervous system serve electrical signaling between neurons, thus they are also called electrical synapses. Such electrical synapses are particularly abundant in the vertebrate retina where they are specialized to form links between neurons as well as glial cells. In this article, we summarize recent findings on retinal cell-to-cell coupling in different vertebrates and identify general features in the light of the evergrowing body of data. In particular, we describe and discuss tracer coupling patterns, connexin proteins, junctional conductances and modulatory processes. This multispecies comparison serves to point out that most features are remarkably conserved across the vertebrate classes, including (i) the cell types connected via electrical synapses; (ii) the connexin makeup and the conductance of each cell-to-cell contact; (iii) the probable function of each gap junction in retinal circuitry; (iv) the fact that gap junctions underlie both electrical and/or tracer coupling between glial cells. These pan-vertebrate features thus demonstrate that retinal gap junctions have changed little during the over 500 million years of vertebrate evolution. Therefore, the fundamental architecture of electrically coupled retinal circuits seems as old as the retina itself, indicating that gap junctions deeply incorporated in retinal wiring from the very beginning of the eye formation of vertebrates. In addition to hard wiring provided by fast synaptic transmitter-releasing neurons and soft wiring contributed by peptidergic, aminergic and purinergic systems, electrical coupling may serve as the 'skeleton' of lateral processing, enabling important functions such

  2. LRP6 acts as a scaffold protein in cardiac gap junction assembly

    PubMed Central

    Li, Jun; Li, Changming; Liang, Dandan; Lv, Fei; Yuan, Tianyou; The, Erlinda; Ma, Xiue; Wu, Yahan; Zhen, Lixiao; Xie, Duanyang; Wang, Shiyi; Liu, Yuan; Huang, Jian; Shi, Jingyi; Liu, Yi; Shi, Dan; Xu, Liang; Lin, Li; Peng, Luying; Cui, Jianmin; Zhu, Weidong; Chen, Yi-Han

    2016-01-01

    Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/β-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/β-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking. PMID:27250245

  3. Gap and tight junctions in the formation of feather branches: A descriptive ultrastructural study.

    PubMed

    Alibardi, Lorenzo

    2010-08-20

    The present study has focused on the distribution and ultrastructure of gap and tight junctions responsible for the formation of the barb/barbule branching in developing feathers using immunocytochemical detection. Apart from desmosomes, both tight and gap junctions are present between differentiating barb/barbule cells and during keratinization. While gap junctions are rare along the perimeter of these cells, tight junctions tend to remain localized in nodes joining barbule cells and between barb cells of the ramus. Occludin and connexin-26 but not connexin-43 have been detected between barb medullary, barb cortical and barbule cells during formation of barbs. Gap junctions are present in supportive cells located in the vicinity of barbule cells and destined to degenerate, but no close junctions are present between supportive and barb/barbule cells. Close junctions mature into penta-laminar junctions that are present between mature barb/barbule cells. Immunolabeling for occludin and Cx26 is rare along these cornified junctions. The junctions allow barb/barbule cells to remain connected until feather-keratin form the mature corneous syncytium that constitutes the barbs. A discussion of the role of gap and tight junctions during feather morphogenesis is presented. 2010 Elsevier GmbH. All rights reserved.

  4. Berberine potentizes apoptosis induced by X-rays irradiation probably through modulation of gap junctions.

    PubMed

    Liu, Bing; Wang, Qin; Yuan, Dong-dong; Hong, Xiao-ting; Tao, Liang

    2011-04-01

    Clinical combination of some traditional Chinese medical herbs, including berberine, with irradiation is demonstrated to improve efficacy of tumor radiotherapy, yet the mechanisms for such effect remain largely unknown. The present study investigated the effect of berberine on apoptosis induced by X-rays irradiation and the relation between this effect and gap junction intercellular communication (GJIC). The role of gap junctions in the modulation of X-rays irradiation-induced apoptosis was explored by manipulation of connexin (Cx) expression, and gap junction function, using oleamide, a GJIC inhibitor, and berberine. In transfected HeLa cells, Cx32 expression increased apoptosis induced by X-rays irradiation, while inhibition of gap junction by oleamide reduced the irradiation responses, indicating the dependence of X-rays irradiation-induced apoptosis on GJIC. Berberine, at the concentrations without cytotoxicity, enhanced apoptosis induced by irradiation only in the presence of functional gap junctions. These results suggest that berberine potentizes cell apoptosis induced by X-rays irradiation, probably through enhancement of gap junction activity.

  5. Role of Non-Myocyte Gap Junctions and Connexin Hemichannels in Cardiovascular Health and Disease: Novel Therapeutic Targets?

    PubMed

    Johnson, Robert D; Camelliti, Patrizia

    2018-03-15

    The heart is a complex organ composed of multiple cell types, including cardiomyocytes and different non-myocyte populations, all working closely together to determine the hearts properties and maintain normal cardiac function. Connexins are abundantly expressed proteins that form plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular channels that allow for communication between cells, and in the heart they play a crucial role in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages. Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this review is to provide insight into connexin-based signalling in non-myocytes during health and disease, and highlight how targeting these proteins could lead to the development of novel therapies. We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular remodelling following myocardial infarction, and are associated with the initiation and development of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting new research avenue with great potential.

  6. Osmotic forces and gap junctions in spreading depression: a computational model

    NASA Technical Reports Server (NTRS)

    Shapiro, B. E.

    2001-01-01

    In a computational model of spreading depression (SD), ionic movement through a neuronal syncytium of cells connected by gap junctions is described electrodiffusively. Simulations predict that SD will not occur unless cells are allowed to expand in response to osmotic pressure gradients and K+ is allowed to move through gap junctions. SD waves of [K+]out approximately 25 to approximately 60 mM moving at approximately 2 to approximately 18 mm/min are predicted over the range of parametric values reported in gray matter, with extracellular space decreasing up to approximately 50%. Predicted waveform shape is qualitatively similar to laboratory reports. The delayed-rectifier, NMDA, BK, and Na+ currents are predicted to facilitate SD, while SK and A-type K+ currents and glial activity impede SD. These predictions are consonant with recent findings that gap junction poisons block SD and support the theories that cytosolic diffusion via gap junctions and osmotic forces are important mechanisms underlying SD.

  7. Managing the complexity of communication: regulation of gap junctions by post-translational modification

    PubMed Central

    Axelsen, Lene N.; Calloe, Kirstine; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten S.

    2013-01-01

    Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720

  8. [Gap junctions: A new therapeutic target in major depressive disorder?].

    PubMed

    Sarrouilhe, D; Dejean, C

    2015-11-01

    Major depressive disorder is a multifactorial chronic and debilitating mood disease with high lifetime prevalence and is associated with excess mortality, especially from cardiovascular diseases and through suicide. The treatments of this disease with tricyclic antidepressants and monoamine oxidase inhibitors are poorly tolerated and those that selectively target serotonin and norepinephrine re-uptake are not effective in all patients, showing the need to find new therapeutic targets. Post-mortem studies of brains from patients with major depressive disorders described a reduced expression of the gap junction-forming membrane proteins connexin 30 and connexin 43 in the prefrontal cortex and the locus coeruleus. The use of chronic unpredictable stress, a rodent model of depression, suggests that astrocytic gap junction dysfunction contributes to the pathophysiology of major depressive disorder. Chronic treatments of rats with fluoxetine and of rat cultured cortical astrocytes with amitriptyline support the hypothesis that the upregulation of gap junctional intercellular communication between brain astrocytes could be a novel mechanism for the therapeutic effect of antidepressants. In conclusion, astrocytic gap junctions are emerging as a new potential therapeutic target for the treatment of patients with major depressive disorder. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  9. Effects of vitamin D receptor knockout on cornea epithelium gap junctions.

    PubMed

    Lu, Xiaowen; Watsky, Mitchell A

    2014-05-06

    Gap junctions are present in all corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different cornea epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact corneas. Previous gap junction studies in cornea epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the corneal epithelium phenotypes and of keratocytes, residing in intact corneas, were detected using FRAP. Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial epithelium.

  10. Ephaptic coupling rescues conduction failure in weakly coupled cardiac tissue with voltage-gated gap junctions

    NASA Astrophysics Data System (ADS)

    Weinberg, S. H.

    2017-09-01

    Electrical conduction in cardiac tissue is usually considered to be primarily facilitated by gap junctions, providing a pathway between the intracellular spaces of neighboring cells. However, recent studies have highlighted the role of coupling via extracellular electric fields, also known as ephaptic coupling, particularly in the setting of reduced gap junction expression. Further, in the setting of reduced gap junctional coupling, voltage-dependent gating of gap junctions, an oft-neglected biophysical property in computational studies, produces a positive feedback that promotes conduction failure. We hypothesized that ephaptic coupling can break the positive feedback loop and rescue conduction failure in weakly coupled cardiac tissue. In a computational tissue model incorporating voltage-gated gap junctions and ephaptic coupling, we demonstrate that ephaptic coupling can rescue conduction failure in weakly coupled tissue. Further, ephaptic coupling increased conduction velocity in weakly coupled tissue, and importantly, reduced the minimum gap junctional coupling necessary for conduction, most prominently at fast pacing rates. Finally, we find that, although neglecting gap junction voltage-gating results in negligible differences in well coupled tissue, more significant differences occur in weakly coupled tissue, greatly underestimating the minimal gap junctional coupling that can maintain conduction. Our study suggests that ephaptic coupling plays a conduction-preserving role, particularly at rapid heart rates.

  11. Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans

    PubMed Central

    Jang, Heeun; Levy, Sagi; Flavell, Steven W.; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I.

    2017-01-01

    A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans. The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9–containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9–based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits. PMID:28143932

  12. Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.

    PubMed

    Jang, Heeun; Levy, Sagi; Flavell, Steven W; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I

    2017-02-14

    A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9 -based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

  13. Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

    PubMed Central

    Pilarczyk, Götz; Nesnidal, Ines; Gunkel, Manuel; Bach, Margund; Bestvater, Felix; Hausmann, Michael

    2017-01-01

    In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin-1β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ-irradiation or alternatively neuregulin-1β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation. PMID:28208769

  14. Role of Gap Junctions in Early Brain Injury Following Subarachnoid Hemorrhage

    PubMed Central

    Ayer, Robert; Chen, Wanqiu; Sugawara, Takashi; Suzuki, Hidenori; Zhang, John H.

    2010-01-01

    Gap junction inhibition has been demonstrated to reverse the vascular contraction that follows experimental subarachnoid hemorrhage. This study hypothesizes that the use of established gap junction inhibitors: octonal and carbenoxolone, to interrupt cell to cell communication will provide neuroprotection against early brain injury after SAH. The filament perforation model of SAH was performed in male Sprague–Dawley rats weighing between 300 and 380g. Octanol (260.46mg or 781.38 mg/kg), carbenoxolone (100 mg/kg), or vehicles were given via intraperitoneal injection 1 hour after SAH. Neurologic deficits and cerebral apoptosis were assessed 24 and 72 hours after SAH. In addition, Western blot analysis was performed to confirm the in vivo inhibition of CNS gap junctions. The administration of octanol and carbenoxolone both failed to attenuate the neurological deficits induced by SAH, and they did not reduce neuronal apoptosis. Additionally, carbenoloxone increased post SAH mortality and exacerbated SAH induced apoptosis. Despites previous studies that show gap junction inhibitors reverse vasospasm following experimental SAH, they failed to improve clinical outcomes or provide neuroprotection in this study. PMID:20018179

  15. Analysis of four connexin26 mutant gap junctions and hemichannels reveals variations in hexamer stability.

    PubMed

    Ambrosi, Cinzia; Boassa, Daniela; Pranskevich, Jennifer; Smock, Amy; Oshima, Atsunori; Xu, Ji; Nicholson, Bruce J; Sosinsky, Gina E

    2010-05-19

    Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Gap junction blockade induces apoptosis in human endometrial stromal cells.

    PubMed

    Yu, Jie; Berga, Sarah L; Zou, Wei; Sun, He-Ying; Johnston-MacAnanny, Erika; Yalcinkaya, Tamer; Sidell, Neil; Bagchi, Indrani C; Bagchi, Milan K; Taylor, Robert N

    2014-07-01

    One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies. © 2014 Wiley Periodicals, Inc.

  17. Meclofenamic acid blocks the gap junction communication between the retinal pigment epithelial cells.

    PubMed

    Ning, N; Wen, Y; Li, Y; Li, J

    2013-11-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to manage the pain and inflammation. NSAIDs can cause serious side effects, including vision problems. However, the underlying mechanisms are still unclear. Therefore, we aimed to investigate the effect of meclofenamic acid (MFA) on retinal pigment epithelium (RPE). In our study, we applied image analysis and whole-cell patch clamp recording to directly measure the effect of MFA on the gap junctional coupling between RPE cells. Analysis of Lucifer yellow (LY) transfer revealed that the gap junction communication existed between RPE cells. Functional experiments using the whole-cell configuration of the patch clamp technique showed that a gap junction conductance also existed between this kind of cells. Importantly, MFA largely inhibited the gap junction conductance and induced the uncoupling of RPE cells. Other NSAIDs, like aspirin and flufenamic acid (FFA), had the same effect. The gap junction functionally existed in RPE cells, which can be blocked by MFA. These findings may explain, at least partially, the vision problems with certain clinically used NSAIDs.

  18. Broad gap junction blocker carbenoxolone disrupts uterine preparation for embryo implantation in mice.

    PubMed

    Diao, Honglu; Xiao, Shuo; Howerth, Elizabeth W; Zhao, Fei; Li, Rong; Ard, Mary B; Ye, Xiaoqin

    2013-08-01

    Gap junctions have an important role in cell-to-cell communication, a process obviously required for embryo implantation. Uterine luminal epithelium (LE) is the first contact for an implanting embryo and is critical for the establishment of uterine receptivity. Microarray analysis of the LE from peri-implantation mouse uterus showed low-level expression of 19 gap junction proteins in preimplantation LE and upregulation of gap junction protein, beta 2 (GJB2, connexin 26, Cx26) in postimplantation LE. Time course study using in situ hybridization and immunofluorescence revealed upregulation of GJB2 in the LE surrounding the implantation site before decidualization. Similar dynamic expression of GJB2 was observed in the LE of artificially decidualized mice but not pseudopregnant mice. To determine the potential function of uterine gap junctions in embryo implantation, carbenoxolone (CBX), a broad gap junction blocker, was injected i.p. (100 mg/kg) or via local uterine fat pad (10 mg/kg) into pregnant mice on Gestation Day 3 at 1800 h, a few hours before embryo attachment to the LE. These CBX treatments disrupted embryo implantation, suggesting local effects of CBX in the uterus. However, i.p. injection of glycyrrhizic acid (100 mg/kg), which shares similar structure and multiple properties with CBX but is ineffective in blocking gap junctions, did not affect embryo implantation. Carbenoxolone also inhibited oil-induced artificial decidualization, concomitant with suppressed molecular changes and ultrastructural transformations associated with uterine preparation for embryo implantation, underscoring the adverse effect of CBX on uterine preparation for embryo implantation. These data demonstrate that uterine gap junctions are important for embryo implantation.

  19. Bioglass promotes wound healing by affecting gap junction connexin 43 mediated endothelial cell behavior.

    PubMed

    Li, Haiyan; He, Jin; Yu, Hongfei; Green, Colin R; Chang, Jiang

    2016-04-01

    It is well known that gap junctions play an important role in wound healing, and bioactive glass (BG) has been shown to help healing when applied as a wound dressing. However, the effects of BG on gap junctional communication between cells involved in wound healing is not well understood. We hypothesized that BG may be able to affect gap junction mediated cell behavior to enhance wound healing. Therefore, we set out to investigate the effects of BG on gap junction related behavior of endothelial cells in order to elucidate the mechanisms through which BG is operating. In in vitro studies, BG ion extracts prevented death of human umbilical vein endothelial cells (HUVEC) following hypoxia in a dose dependent manner, possibly through connexin hemichannel modulation. In addition, BG showed stimulatory effects on gap junction communication between HUVECs and upregulated connexin43 (Cx43) expression. Furthermore, BG prompted expression of vascular endothelial growth factor and basic fibroblast growth factor as well as their receptors, and vascular endothelial cadherin in HUVECs, all of which are beneficial for vascularization. In vivo wound healing results showed that the wound closure of full-thickness excisional wounds of rats was accelerated by BG with reduced inflammation during initial stages of healing and stimulated angiogenesis during the proliferation stage. Therefore, BG can stimulate wound healing through affecting gap junctions and gap junction related endothelial cell behaviors, including prevention of endothelial cell death following hypoxia, stimulation of gap junction communication and upregulation of critical vascular growth factors, which contributes to the enhancement of angiogenesis in the wound bed and finally to accelerate wound healing. Although many studies have reported that BG stimulates angiogenesis and wound healing, this work reveals the relationship between BG and gap junction connexin 43 mediated endothelial cell behavior and elucidates

  20. The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain.

    PubMed

    Li, X; Lynn, B D; Nagy, J I

    2012-01-01

    Electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36) occur in most major structures in the mammalian central nervous system. These synapses link ensembles of neurons and influence their network properties. Little is known about the macromolecular constituents of neuronal gap junctions or how transmission through electrical synapses is regulated at the level of channel conductance or gap junction assembly/disassembly. Such knowledge is a prerequisite to understanding the roles of gap junctions in neuronal circuitry. Gap junctions share similarities with tight and adhesion junctions in that all three reside at close plasma membrane appositions, and therefore may associate with similar structural and regulatory proteins. Previously, we reported that the tight junction-associated protein zonula occludens-1 (ZO-1) interacts with Cx36 and is localized at gap junctions. Here, we demonstrate that two proteins known to be associated with tight and adherens junctions, namely AF6 and MUPP1, are components of neuronal gap junctions in rodent brain. By immunofluorescence, AF6 and MUPP1 were co-localized with Cx36 in many brain areas. Co-immunoprecipitation and pull-down approaches revealed an association of Cx36 with AF6 and MUPP1, which required the C-terminus PDZ domain interaction motif of Cx36 for interaction with the single PDZ domain of AF6 and with the 10th PDZ domain of MUPP1. As AF6 is a target of the cAMP/Epac/Rap1 signalling pathway and MUPP1 is a scaffolding protein that interacts with CaMKII, the present results suggest that AF6 may be a target for cAMP/Epac/Rap1 signalling at electrical synapses, and that MUPP1 may contribute to anchoring CaMKII at these synapses. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  1. Gap Junctions in the Ventral Hippocampal-Medial Prefrontal Pathway Are Involved in Anxiety Regulation

    PubMed Central

    Schoenfeld, Timothy J.; Kloth, Alexander D.; Hsueh, Brian; Runkle, Matthew B.; Kane, Gary A.; Wang, Samuel S.-H.

    2014-01-01

    Anxiety disorders are highly prevalent but little is known about their underlying mechanisms. Gap junctions exist in brain regions important for anxiety regulation, such as the ventral hippocampus (vHIP) and mPFC, but their functions in these areas have not been investigated. Using pharmacological blockade of neuronal gap junctions combined with electrophysiological recordings, we found that gap junctions play a role in theta rhythm in the vHIP and mPFC of adult mice. Bilateral infusion of neuronal gap junction blockers into the vHIP decreased anxiety-like behavior on the elevated plus maze and open field. Similar anxiolytic effects were observed with unilateral infusion of these drugs into the vHIP combined with contralateral infusion into the mPFC. No change in anxious behavior was observed with gap junction blockade in the unilateral vHIP alone or in the bilateral dorsal HIP. Since physical exercise is known to reduce anxiety, we examined the effects of long-term running on the expression of the neuronal gap junction protein connexin-36 among inhibitory interneurons and found a reduction in the vHIP. Despite this change, we observed no alteration in theta frequency or power in long-term runners. Collectively, these findings suggest that neuronal gap junctions in the vHIP–mPFC pathway are important for theta rhythm and anxiety regulation under sedentary conditions but that additional mechanisms are likely involved in running-induced reduction in anxiety. PMID:25411496

  2. Involvement of connexin 43 phosphorylation and gap junctional communication between smooth muscle cells in vasopressin-induced ROCK-dependent vasoconstriction after hemorrhagic shock.

    PubMed

    Yang, Guangming; Peng, Xiaoyong; Wu, Yue; Li, Tao; Liu, Liangming

    2017-10-01

    We examined the roles played by gap junctions (GJs) and the GJ channel protein connexin 43 (Cx43) in arginine vasopressin (AVP)-induced vasoconstriction after hemorrhagic shock and their relationship to Rho kinase (ROCK) and protein kinase C (PKC). The results showed that AVP induced an endothelium-independent contraction in rat superior mesenteric arteries (SMAs). Blocking the GJs significantly decreased the contractile response of SMAs and vascular smooth muscle cells (VSMCs) to AVP after shock and hypoxia. The selective Cx43-mimetic peptide inhibited the vascular contractile effect of AVP after shock and hypoxia. AVP restored hypoxia-induced decrease of Cx43 phosphorylation at Ser 262 and gap junctional communication in VSMCs. Activation of RhoA with U-46619 increased the contractile effect of AVP. This effect was antagonized by the ROCK inhibitor Y27632 and the Cx43-mimetic peptide. In contrast, neither an agonist nor an inhibitor of PKC had significant effects on AVP-induced contraction after hemorrhagic shock. In addition, silencing of Cx43 with siRNA blocked the AVP-induced increase of ROCK activity in hypoxic VSMCs. In conclusion, AVP-mediated vascular contractile effects are endothelium and myoendothelial gap junction independent. Gap junctions between VSMCs, gap junctional communication, and Cx43 phosphorylation at Ser 262 play important roles in the vascular effects of AVP. RhoA/ROCK, but not PKC, is involved in this process. Copyright © 2017 the American Physiological Society.

  3. Gap junction disorders of myelinating cells.

    PubMed

    Kleopa, Kleopas A; Orthmann-Murphy, Jennifer; Sargiannidou, Irene

    2010-01-01

    Gap junctions (GJs) are channels that allow the diffusion of ions and small molecules across apposed cell membranes. In peripheral nerves, Schwann cells express the GJ proteins connexin32 (Cx32) and Cx29, which have distinct localizations. Cx32 forms GJs through non-compact myelin areas, whereas Cx29 forms hemichannels in the innermost layers of myelin apposing axonal Shaker-type K+ channels. In the CNS, rodent oligodendrocytes express Cx47, Cx32 and Cx29. Cx47 is expressed by all types of oligodendrocytes both in the white and grey matter and forms GJs on cell bodies and proximal processes, as well as most of the intercellular channels with astrocytes. Cx32 is expressed mostly by white matter oligodendrocytes and is localized in the myelin sheath of large diameter fibers. Cx29, and its human ortholog Cx31.3, appear to be restricted to oligodendrocytes that myelinate small caliber fibers, likely forming hemichannels. The importance of intercellular and intracellular GJs in myelinating cells are demonstrated by human disorders resulting from mutations affecting GJ proteins. The X-linked Charcot Marie Tooth disease (CMT1X) is caused by hundreds of mutations affecting Cx32. Patients with CMT1X present mainly with a progressive peripheral neuropathy, which may be accompanied by CNS myelin dysfunction. Mutations in Cx47 may cause a devastating leukodystrophy called Pelizaeus-Merzbacher-like disease or a milder spastic paraplegia. In addition, CNS demyelination may be caused by defects in genes expressing astrocytic GJ proteins, which are essential for oligodendrocytes. Findings from in vitro and in vivo models of these disorders developed over the last decade indicate that most mutations cause loss of function and an inability of the mutant connexins to form functional GJs. Here we review the clinical, genetic, and neurobiological aspects of GJ disorders affecting the PNS and CNS myelinating cells.

  4. Voltage-Controlled Switching and Thermal Effects in VO2 Nano-Gap Junctions

    DTIC Science & Technology

    2014-06-09

    Voltage-controlled switching and thermal effects in VO2 nano-gap junctions Arash Joushaghani,1 Junho Jeong,1 Suzanne Paradis,2 David Alain,2 J...2014) Voltage-controlled switching in lateral VO2 nano-gap junctions with different gap lengths and thermal properties was investigated. The effect of...indicate that the VO2 phase transition was likely initiated electroni- cally, which was sometimes followed by a secondary thermally-induced transition

  5. Structural analysis of key gap junction domains--Lessons from genome data and disease-linked mutants.

    PubMed

    Bai, Donglin

    2016-02-01

    A gap junction (GJ) channel is formed by docking of two GJ hemichannels and each of these hemichannels is a hexamer of connexins. All connexin genes have been identified in human, mouse, and rat genomes and their homologous genes in many other vertebrates are available in public databases. The protein sequences of these connexins align well with high sequence identity in the same connexin across different species. Domains in closely related connexins and several residues in all known connexins are also well-conserved. These conserved residues form signatures (also known as sequence logos) in these domains and are likely to play important biological functions. In this review, the sequence logos of individual connexins, groups of connexins with common ancestors, and all connexins are analyzed to visualize natural evolutionary variations and the hot spots for human disease-linked mutations. Several gap junction domains are homologous, likely forming similar structures essential for their function. The availability of a high resolution Cx26 GJ structure and the subsequently-derived homology structure models for other connexin GJ channels elevated our understanding of sequence logos at the three-dimensional GJ structure level, thus facilitating the understanding of how disease-linked connexin mutants might impair GJ structure and function. This knowledge will enable the design of complementary variants to rescue disease-linked mutants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Tight junctions of the proximal tubule and their channel proteins.

    PubMed

    Fromm, Michael; Piontek, Jörg; Rosenthal, Rita; Günzel, Dorothee; Krug, Susanne M

    2017-08-01

    The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na + and K + . Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na + and water share the same pore. Already solved claudin crystal structures reveal a characteristic β-sheet, comprising β-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

  7. Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart.

    PubMed

    Palatinus, Joseph A; Gourdie, Robert G

    2016-01-01

    Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43) in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

  8. Serotonin passes through myoendothelial gap junctions to promote pulmonary arterial smooth muscle cell differentiation.

    PubMed

    Gairhe, Salina; Bauer, Natalie N; Gebb, Sarah A; McMurtry, Ivan F

    2012-11-01

    Myoendothelial gap junctional signaling mediates pulmonary arterial endothelial cell (PAEC)-induced activation of latent TGF-β and differentiation of cocultured pulmonary arterial smooth muscle cells (PASMCs), but the nature of the signal passing from PAECs to PASMCs through the gap junctions is unknown. Because PAECs but not PASMCs synthesize serotonin, and serotonin can pass through gap junctions, we hypothesized that the monoamine is the intercellular signal. We aimed to determine whether PAEC-derived serotonin mediates PAEC-induced myoendothelial gap junction-dependent activation of TGF-β signaling and differentiation of PASMCs. Rat PAECs and PASMCs were monocultured or cocultured with (touch) or without (no-touch) direct cell-cell contact. In all cases, tryptophan hydroxylase 1 (Tph1) transcripts were expressed predominantly in PAECs. Serotonin was detected by immunostaining in both PAECs and PASMCs in PAEC/PASMC touch coculture but was not found in PASMCs in either PAEC/PASMC no-touch coculture or in PASMC/PASMC touch coculture. Furthermore, inhibition of gap junctions but not of the serotonin transporter in PAEC/PASMC touch coculture prevented serotonin transfer from PAECs to PASMCs. Inhibition of serotonin synthesis pharmacologically or by small interfering RNAs to Tph1 in PAECs inhibited the PAEC-induced activation of TGF-β signaling and differentiation of PASMCs. We concluded that serotonin synthesized by PAECs is transferred through myoendothelial gap junctions into PASMCs, where it activates TGF-β signaling and induces a more differentiated phenotype. This finding suggests a novel role of gap junction-mediated intercellular serotonin signaling in regulation of PASMC phenotype.

  9. Thalamic modulation of cingulate seizure activity via the regulation of gap junctions in mice thalamocingulate slice.

    PubMed

    Chang, Wei-Pang; Wu, José Jiun-Shian; Shyu, Bai-Chuang

    2013-01-01

    The thalamus is an important target for deep brain stimulation in the treatment of seizures. However, whether the modulatory effect of thalamic inputs on cortical seizures occurs through the modulation of gap junctions has not been previously studied. Therefore, we tested the effects of different gap junction blockers and couplers in a drug-resistant seizure model and studied the role of gap junctions in the thalamic modulation on cortical seizures. Multielectrode array and calcium imaging were used to record the cortical seizures induced by 4-aminopyridine (250 µM) and bicuculline (5-50 µM) in a novel thalamocingulate slice preparation. Seizure-like activity was significantly attenuated by the pan-gap junction blockers carbenoxolone and octanol and specific neuronal gap junction blocker mefloquine. The gap junction coupler trimethylamine significantly enhanced seizure-like activity. Gap junction blockers did not influence the initial phase of seizure-like activity, but they significantly decreased the amplitude and duration of the maintenance phase. The development of seizures is regulated by extracellular potassium concentration. Carbenoxolone partially restored the amplitude and duration after removing the thalamic inputs. A two-dimensional current source density analysis showed that the sink and source signals shifted to deeper layers after removing the thalamic inputs during the clonic phase. These results indicate that the regulatory mechanism of deep brain stimulation in the thalamus occurs partially though gap junctions.

  10. Thalamic Modulation of Cingulate Seizure Activity Via the Regulation of Gap Junctions in Mice Thalamocingulate Slice

    PubMed Central

    Chang, Wei-Pang; Wu, José Jiun-Shian; Shyu, Bai-Chuang

    2013-01-01

    The thalamus is an important target for deep brain stimulation in the treatment of seizures. However, whether the modulatory effect of thalamic inputs on cortical seizures occurs through the modulation of gap junctions has not been previously studied. Therefore, we tested the effects of different gap junction blockers and couplers in a drug-resistant seizure model and studied the role of gap junctions in the thalamic modulation on cortical seizures. Multielectrode array and calcium imaging were used to record the cortical seizures induced by 4-aminopyridine (250 µM) and bicuculline (5–50 µM) in a novel thalamocingulate slice preparation. Seizure-like activity was significantly attenuated by the pan-gap junction blockers carbenoxolone and octanol and specific neuronal gap junction blocker mefloquine. The gap junction coupler trimethylamine significantly enhanced seizure-like activity. Gap junction blockers did not influence the initial phase of seizure-like activity, but they significantly decreased the amplitude and duration of the maintenance phase. The development of seizures is regulated by extracellular potassium concentration. Carbenoxolone partially restored the amplitude and duration after removing the thalamic inputs. A two-dimensional current source density analysis showed that the sink and source signals shifted to deeper layers after removing the thalamic inputs during the clonic phase. These results indicate that the regulatory mechanism of deep brain stimulation in the thalamus occurs partially though gap junctions. PMID:23690968

  11. Communication-dependent mineralization of osteoblasts via gap junctions.

    PubMed

    Hashida, Yukihiko; Nakahama, Ken-ichi; Shimizu, Kaori; Akiyama, Masako; Harada, Kiyoshi; Morita, Ikuo

    2014-04-01

    Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Myosin VI facilitates connexin 43 gap junction accretion.

    PubMed

    Waxse, Bennett J; Sengupta, Prabuddha; Hesketh, Geoffrey G; Lippincott-Schwartz, Jennifer; Buss, Folma

    2017-03-01

    In this study, we demonstrate myosin VI enrichment at Cx43 (also known as GJA1)-containing gap junctions (GJs) in heart tissue, primary cardiomyocytes and cell culture models. In primary cardiac tissue and in fibroblasts from the myosin VI-null mouse as well as in tissue culture cells transfected with siRNA against myosin VI, we observe reduced GJ plaque size with a concomitant reduction in intercellular communication, as shown by fluorescence recovery after photobleaching (FRAP) and a new method of selective calcein administration. Analysis of the molecular role of myosin VI in Cx43 trafficking indicates that myosin VI is dispensable for the delivery of Cx43 to the cell surface and connexon movement in the plasma membrane. Furthermore, we cannot corroborate clathrin or Dab2 localization at gap junctions and we do not observe a function for the myosin-VI-Dab2 complex in clathrin-dependent endocytosis of annular gap junctions. Instead, we found that myosin VI was localized at the edge of Cx43 plaques by using total internal reflection fluorescence (TIRF) microscopy and use FRAP to identify a plaque accretion defect as the primary manifestation of myosin VI loss in Cx43 homeostasis. A fuller understanding of this derangement may explain the cardiomyopathy or gliosis associated with the loss of myosin VI. © 2017. Published by The Company of Biologists Ltd.

  13. Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

    NASA Astrophysics Data System (ADS)

    Takaku, Yasuharu; Hwang, Jung Shan; Wolf, Alexander; Böttger, Angelika; Shimizu, Hiroshi; David, Charles N.; Gojobori, Takashi

    2014-01-01

    Nerve cells and spontaneous coordinated behavior first appeared near the base of animal evolution in the common ancestor of cnidarians and bilaterians. Experiments on the cnidarian Hydra have demonstrated that nerve cells are essential for this behavior, although nerve cells in Hydra are organized in a diffuse network and do not form ganglia. Here we show that the gap junction protein innexin-2 is expressed in a small group of nerve cells in the lower body column of Hydra and that an anti-innexin-2 antibody binds to gap junctions in the same region. Treatment of live animals with innexin-2 antibody eliminates gap junction staining and reduces spontaneous body column contractions. We conclude that a small subset of nerve cells, connected by gap junctions and capable of synchronous firing, act as a pacemaker to coordinate the contraction of the body column in the absence of ganglia.

  14. Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

    PubMed Central

    Takaku, Yasuharu; Hwang, Jung Shan; Wolf, Alexander; Böttger, Angelika; Shimizu, Hiroshi; David, Charles N.; Gojobori, Takashi

    2014-01-01

    Nerve cells and spontaneous coordinated behavior first appeared near the base of animal evolution in the common ancestor of cnidarians and bilaterians. Experiments on the cnidarian Hydra have demonstrated that nerve cells are essential for this behavior, although nerve cells in Hydra are organized in a diffuse network and do not form ganglia. Here we show that the gap junction protein innexin-2 is expressed in a small group of nerve cells in the lower body column of Hydra and that an anti-innexin-2 antibody binds to gap junctions in the same region. Treatment of live animals with innexin-2 antibody eliminates gap junction staining and reduces spontaneous body column contractions. We conclude that a small subset of nerve cells, connected by gap junctions and capable of synchronous firing, act as a pacemaker to coordinate the contraction of the body column in the absence of ganglia. PMID:24394722

  15. Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.

    PubMed

    Takaku, Yasuharu; Hwang, Jung Shan; Wolf, Alexander; Böttger, Angelika; Shimizu, Hiroshi; David, Charles N; Gojobori, Takashi

    2014-01-07

    Nerve cells and spontaneous coordinated behavior first appeared near the base of animal evolution in the common ancestor of cnidarians and bilaterians. Experiments on the cnidarian Hydra have demonstrated that nerve cells are essential for this behavior, although nerve cells in Hydra are organized in a diffuse network and do not form ganglia. Here we show that the gap junction protein innexin-2 is expressed in a small group of nerve cells in the lower body column of Hydra and that an anti-innexin-2 antibody binds to gap junctions in the same region. Treatment of live animals with innexin-2 antibody eliminates gap junction staining and reduces spontaneous body column contractions. We conclude that a small subset of nerve cells, connected by gap junctions and capable of synchronous firing, act as a pacemaker to coordinate the contraction of the body column in the absence of ganglia.

  16. Antidromic-rectifying gap junctions amplify chemical transmission at functionally mixed electrical-chemical synapses

    PubMed Central

    Liu, Ping; Chen, Bojun; Mailler, Roger; Wang, Zhao-Wen

    2017-01-01

    Neurons communicate through chemical synapses and electrical synapses (gap junctions). Although these two types of synapses often coexist between neurons, little is known about whether they interact, and whether any interactions between them are important to controlling synaptic strength and circuit functions. By studying chemical and electrical synapses between premotor interneurons (AVA) and downstream motor neurons (A-MNs) in the Caenorhabditis elegans escape circuit, we found that disrupting either the chemical or electrical synapses causes defective escape response. Gap junctions between AVA and A-MNs only allow antidromic current, but, curiously, disrupting them inhibits chemical transmission. In contrast, disrupting chemical synapses has no effect on the electrical coupling. These results demonstrate that gap junctions may serve as an amplifier of chemical transmission between neurons with both electrical and chemical synapses. The use of antidromic-rectifying gap junctions to amplify chemical transmission is potentially a conserved mechanism in circuit functions. PMID:28317880

  17. Oxaliplatin-induced neurotoxicity is mediated through gap junction channels and hemichannels and can be prevented by octanol.

    PubMed

    Kagiava, Alexia; Theophilidis, George; Sargiannidou, Irene; Kyriacou, Kyriacos; Kleopa, Kleopas A

    2015-10-01

    Oxaliplatin-induced neurotoxicity (OIN) is a common complication of chemotherapy without effective treatment. In order to clarify the mechanisms of both acute and chronic OIN, we used an ex-vivo mouse sciatic nerve model. Exposure to 25 μM oxaliplatin caused a marked prolongation in the duration of the nerve evoked compound action potential (CAP) by nearly 1200% within 300 min while amplitude remained constant for over 20 h. This oxaliplatin effect was almost completely reversed by the gap junction (GJ) inhibitor octanol in a concentration-dependent manner. Further GJ blockers showed similar effects although with a narrower therapeutic window. To clarify the target molecule we studied sciatic nerves from connexin32 (Cx32) and Cx29 knockout (KO) mice. The oxaliplatin effect and neuroprotection by octanol partially persisted in Cx29 better than in Cx32 KO nerves, suggesting that oxaliplatin affects both, but Cx32 GJ channels more than Cx29 hemichannels. Oxaliplatin also accelerated neurobiotin uptake in HeLa cells expressing the human ortholog of Cx29, Cx31.3, as well as dye transfer between cells expressing the human Cx32, and this effect was blocked by octanol. Oxaliplatin caused no morphological changes initially (up to 3 h of exposure), but prolonged nerve exposure caused juxtaparonodal axonal edema, which was prevented by octanol. Our study indicates that oxaliplatin causes forced opening of Cx32 channels and Cx29 hemichannels in peripheral myelinated fibers leading to disruption of axonal K(+) homeostasis. The GJ blocker octanol prevents OIN at very low concentrations and should be further studied as a neuroprotectant. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

    PubMed

    Vliagoftis, Harissios; Ebeling, Cory; Ilarraza, Ramses; Mahmudi-Azer, Salahaddin; Abel, Melanie; Adamko, Darryl; Befus, A Dean; Moqbel, Redwan

    2014-01-01

    Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

  19. Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus).

    PubMed

    Lavrov, Igor; Fox, Lyle; Shen, Jun; Han, Yingchun; Cheng, Jianguo

    2016-01-01

    Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

  20. Terbinafine inhibits gap junctional intercellular communication

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Ju Yeun, E-mail: whitewndus@naver.com

    Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca{sup 2+} concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibitsmore » GJIC with a so far unknown mechanism of action. - Highlights: • In vitro pharmacological studies were performed on FRT-Cx43 and LN215 cells. • Terbinafine inhibits gap junctional intercellular communication in both cell lines. • The inhibitory effect of terbinafine is reversible and dose-dependent. • Treatment of terbinafine does not alter Cx43 phosphorylation or cytosolic Ca{sup 2+} concentration. • Inhibition of squalene epoxidase is not involved in this new effect of terbinafine.« less

  1. Extracellular domains play different roles in gap junction formation and docking compatibility.

    PubMed

    Bai, Donglin; Wang, Ao Hong

    2014-02-15

    GJ (gap junction) channels mediate direct intercellular communication and play an important role in many physiological processes. Six connexins oligomerize to form a hemichannel and two hemichannels dock together end-to-end to form a GJ channel. Connexin extracellular domains (E1 and E2) have been shown to be important for the docking, but the molecular mechanisms behind the docking and formation of GJ channels are not clear. Recent developments in atomic GJ structure and functional studies on a series of connexin mutants revealed that E1 and E2 are likely to play different roles in the docking. Non-covalent interactions at the docking interface, including hydrogen bonds, are predicted to form between interdocked extracellular domains. Protein sequence alignment analysis on the docking compatible/incompatible connexins indicate that the E1 domain is important for the formation of the GJ channel and the E2 domain is important in the docking compatibility in heterotypic channels. Interestingly, the hydrogen-bond forming or equivalent residues in both E1 and E2 domains are mutational hot spots for connexin-linked human diseases. Understanding the molecular mechanisms of GJ docking can assist us to develop novel strategies in rescuing the disease-linked connexin mutants.

  2. Keratitis-Ichthyosis-Deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43

    PubMed Central

    García, Isaac E.; Maripillán, Jaime; Jara, Oscar; Ceriani, Ricardo; Palacios-Muñoz, Angelina; Ramachandran, Jayalakshimi; Olivero, Pablo; Pérez-Acle, Tomás; González, Carlos; Sáez, Juan C.; Contreras, Jorge E.; Martínez, Agustín D.

    2015-01-01

    Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like Keratitis Ichthyosis Deafness syndrome (KID). Because in the human skin Cx26 is co-expressed with other connexins, like Cx43 and Cx30, and since KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channels functions remain unknown. In this study we demonstrate that syndromic mutations at the N-terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) show exacerbated hemichannel activity, but nonfunctional gap junction channels; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca2+ overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin. PMID:25625422

  3. Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity.

    PubMed

    Desforges, Bénédicte; Savarin, Philippe; Bounedjah, Ouissame; Delga, Stéphanie; Hamon, Loïc; Curmi, Patrick A; Pastré, David

    2011-09-01

    Upon hypertonic stress most often resulting from high salinity, cells need to balance their osmotic pressure by accumulating neutral osmolytes called compatible osmolytes like betaine, myo-inositol, and taurine. However, the massive uptake of compatible osmolytes is a slow process compared with other defense mechanisms related to oxidative or heat stress. This is especially critical for cycling cells as they have to double their volume while keeping a hospitable intracellular environment for the molecular machineries. Here we propose that clustered cells can accelerate the supply of compatible osmolytes to cycling cells via the transit, mediated by gap junctions, of compatible osmolytes from arrested to cycling cells. Both experimental results in epithelial normal rat kidney cells and theoretical estimations show that gap junctions indeed play a key role in cell adaptation to chronic hypertonicity. These results can provide basis for a better understanding of the functions of gap junctions in osmoregulation not only for the kidney but also for many other epithelia. In addition to this, we suggest that cancer cells that do not communicate via gap junctions poorly cope with hypertonic environments thus explaining the rare occurrence of cancer coming from the kidney medulla.

  4. Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens.

    PubMed

    Biswas, Sondip K; Lo, Woo-Kuen

    2007-03-09

    To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the embryonic chicken lens. The possible mechanism by which cholesterol is removed from gap junction plaques is also investigated. Filipin cytochemistry in conjunction with freeze-fracture TEM was used to visualize cholesterol, as represented by filipin-cholesterol complexes (FCCs) in gap junction plaques. Quantitative analysis on the heterogeneous distribution of cholesterol in gap junction plaques was conducted from outer and inner cortical regions. A novel technique combining filipin cytochemistry with freeze-fracture replica immunogold labeling (FRIL) was used to label Cx45.6 and Cx56 antibodies in cholesterol-containing gap junctions. Filipin cytochemistry and freeze-fracture TEM and thin-section TEM were used to examine the appearance and nature of the cholesterol-containing vesicular structures associated with gap junction plaques. Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL studies confirmed that cholesterol-containing junctions were gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were often observed in the gap junction plaques. They appear to be involved in the removal of cholesterol from gap junction plaques through endocytosis. Gap junctions in the young fibers are enriched with cholesterol because they are assembled in the unique cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner

  5. High band gap 2-6 and 3-5 tunneling junctions for silicon multijunction solar cells

    NASA Technical Reports Server (NTRS)

    Daud, Taher (Inventor); Kachare, Akaram H. (Inventor)

    1986-01-01

    A multijunction silicon solar cell of high efficiency is provided by providing a tunnel junction between the solar cell junctions to connect them in series. The tunnel junction is comprised of p+ and n+ layers of high band gap 3-5 or 2-6 semiconductor materials that match the lattice structure of silicon, such as GaP (band gap 2.24 eV) or ZnS (band gap 3.6 eV). Each of which has a perfect lattice match with silicon to avoid defects normally associated with lattice mismatch.

  6. Gap-junction-mediated communication in human periodontal ligament cells.

    PubMed

    Kato, R; Ishihara, Y; Kawanabe, N; Sumiyoshi, K; Yoshikawa, Y; Nakamura, M; Imai, Y; Yanagita, T; Fukushima, H; Kamioka, H; Takano-Yamamoto, T; Yamashiro, T

    2013-07-01

    Periodontal tissue homeostasis depends on a complex cellular network that conveys cell-cell communication. Gap junctions (GJs), one of the intercellular communication systems, are found between adjacent human periodontal ligament (hPDL) cells; however, the functional GJ coupling between hPDL cells has not yet been elucidated. In this study, we investigated functional gap-junction-mediated intercellular communication in isolated primary hPDL cells. SEM images indicated that the cells were in contact with each other via dendritic processes, and also showed high anti-connexin43 (Cx43) immunoreactivity on these processes. Gap-junctional intercellular communication (GJIC) among hPDL cells was assessed by fluorescence recovery after a photobleaching (FRAP) analysis, which exhibited dye coupling between hPDL cells, and was remarkably down-regulated when the cells were treated with a GJ blocker. Additionally, we examined GJs under hypoxic stress. The fluorescence recovery and expression levels of Cx43 decreased time-dependently under the hypoxic condition. Exposure to GJ inhibitor or hypoxia increased RANKL expression, and decreased OPG expression. This study shows that GJIC is responsible for hPDL cells and that its activity is reduced under hypoxia. This is consistent with the possible role of hPDL cells in regulating the biochemical reactions in response to changes in the hypoxic environment.

  7. Electron tomographic analysis of gap junctions in lateral giant fibers of crayfish.

    PubMed

    Ohta, Yasumi; Nishikawa, Kouki; Hiroaki, Yoko; Fujiyoshi, Yoshinori

    2011-07-01

    Innexin-gap junctions in crayfish lateral giant fibers (LGFs) have an important role in escape behavior as a key component of rapid signal transduction. Knowledge of the structure and function of characteristic vesicles on the both sides of the gap junction, however, is limited. We used electron tomography to analyze the three-dimensional structure of crayfish gap junctions and gap junctional vesicles (GJVs). Tomographic analyses showed that some vesicles were anchored to innexons and almost all vesicles were connected by thin filaments. High densities inside the GJVs and projecting densities on the GJV membranes were observed in fixed and stained samples. Because the densities inside synaptic vesicles were dependent on the fixative conditions, different fixative conditions were used to elucidate the molecules included in the GJVs. The projecting densities on the GJVs were studied by immunoelectron microscopy with anti-vesicular monoamine transporter (anti-VMAT) and anti-vesicular nucleotide transporter (anti-VNUT) antibodies. Some of the projecting densities were labeled by anti-VNUT, but not anti-VMAT. Three-dimensional analyses of GJVs and excitatory chemical synaptic vesicles (CSVs) revealed clear differences in their sizes and central densities. Furthermore, the imaging data obtained under different fixative conditions and the immunolabeling results, in which GJVs were positively labeled for anti-VNUT but excitatory CSVs were not, support our model that GJVs contain nucleotides and excitatory CSVs do not. We propose a model in which characteristic GJVs containing nucleotides play an important role in the signal processing in gap junctions of crayfish LGFs. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Gap Junction Modulation of Low-Frequency Oscillations in the Cerebellar Granule Cell Layer.

    PubMed

    Robinson, Jennifer Claire; Chapman, C Andrew; Courtemanche, Richard

    2017-08-01

    Local field potential (LFP) oscillations in the granule cell layer (GCL) of the cerebellar cortex have been identified previously in the awake rat and monkey during immobility. These low-frequency oscillations are thought to be generated through local circuit interactions between Golgi cells and granule cells within the GCL. Golgi cells display rhythmic firing and pacemaking properties, and also are electrically coupled through gap junctions within the GCL. Here, we tested if gap junctions in the rat cerebellar cortex contribute to the generation of LFP oscillations in the GCL. We recorded LFP oscillations under urethane anesthesia, and examined the effects of local infusion of gap junction blockers on 5-15 Hz oscillations. Local infusion of the gap junction blockers carbenoxolone and mefloquine resulted in significant decreases in the power of oscillations over a 30-min period, but the power of oscillations was unchanged in control experiments following vehicle injections. In addition, infusion of gap junction blockers had no significant effect on multi-unit activity, suggesting that the attenuation of low-frequency oscillations was likely due to reductions in electrical coupling rather than a decreased excitability within the granule cell layer. Our results indicate that electrical coupling among the Golgi cell networks in the cerebellar cortex contributes to the local circuit mechanisms that promote the occurrence of GCL LFP slow oscillations in the anesthetized rat.

  9. Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

    PubMed

    Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

    2015-11-01

    The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331. © 2015 by

  10. Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

    PubMed

    Raškevičius, Vytautas; Jotautis, Vaidas; Rimkutė, Lina; Marandykina, Alina; Kazokaitė, Mintautė; Kairys, Visvaldas; Skeberdis, Vytenis Arvydas

    2018-02-28

    In our recent study, we have demonstrated that short carbon chain n -alcohols (up to octanol) stimulated while long carbon chain n -alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n -alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n -alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87. © 2018 The Author(s).

  11. Intercellular communication via gap junctions affected by mechanical load in the bovine annulus fibrosus.

    PubMed

    Desrochers, Jane; Duncan, Neil A

    2014-01-01

    Cells in the intervertebral disc, as in other connective tissues including tendon, ligament and bone, form interconnected cellular networks that are linked via functional gap junctions. These cellular networks may be necessary to affect a coordinated response to mechanical and environmental stimuli. Using confocal microscopy with fluorescence recovery after photobleaching methods, we explored the in situ strain environment of the outer annulus of an intact bovine disc and the effect of high-level flexion on gap junction signalling. The in situ strain environment in the extracellular matrix of the outer annulus under high flexion load was observed to be non-uniform with the extensive cellular processes remaining crimped sometimes at flexion angles greater than 25°. A significant transient disruption of intercellular communication via functional gap junctions was measured after 10 and 20 min under high flexion load. This study illustrates that in healthy annulus fibrosus tissue, high mechanical loads can impede the functioning of the gap junctions. Future studies will explore more complex loading conditions to determine whether losses in intercellular communication can be permanent and whether gap junctions in aged and degenerated tissues become more susceptible to load. The current research suggests that cellular structures such as gap junctions and intercellular networks, as well as other cell-cell and cell-matrix interconnections, need to be considered in computational models in order to fully understand how macroscale mechanical signals are transmitted across scales to the microscale and ultimately into a cellular biosynthetic response in collagenous tissues.

  12. Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact.

    PubMed

    Belousov, Andrei B; Wang, Yongfu; Song, Ji-Hoon; Denisova, Janna V; Berman, Nancy E; Fontes, Joseph D

    2012-08-22

    In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death. We report that 24h post-CCI, substantial neuronal death is detected in a number of brain regions outside the injury core, including the striatum. The striatal neuronal death is reduced both in wild-type mice by systemic administration of mefloquine (a relatively selective blocker of neuronal gap junctions) and in knockout mice lacking connexin 36 (neuronal gap junction protein). It is also reduced by inactivation of group II metabotropic glutamate receptors (with LY341495) which, as reported previously, control the rapid increase in neuronal gap junction coupling following different types of neuronal injury. The results suggest that neuronal gap junctions play a critical role in the CCI-induced secondary neuronal death. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  13. Single-junction solar cells with the optimum band gap for terrestrial concentrator applications

    DOEpatents

    Wanlass, Mark W.

    1994-01-01

    A single-junction solar cell having the ideal band gap for terrestrial concentrator applications. Computer modeling studies of single-junction solar cells have shown that the presence of absorption bands in the direct spectrum has the effect of "pinning" the optimum band gap for a wide range of operating conditions at a value of 1.14.+-.0.02 eV. Efficiencies exceeding 30% may be possible at high concentration ratios for devices with the ideal band gap.

  14. Gap junctions between CA3 pyramidal cells contribute to network synchronization in neonatal hippocampus.

    PubMed

    Molchanova, Svetlana M; Huupponen, Johanna; Lauri, Sari E; Taira, Tomi

    2016-08-01

    Direct electrical coupling between neurons through gap junctions is prominent during development, when synaptic connectivity is scarce, providing the additional intercellular connectivity. However, functional studies of gap junctions are hampered by the unspecificity of pharmacological tools available. Here we have investigated gap-junctional coupling between CA3 pyramidal cells in neonatal hippocampus and its contribution to early network activity. Four different gap junction inhibitors, including the general blocker carbenoxolone, decreased the frequency of network activity bursts in CA3 area of hippocampus of P3-6 rats, suggesting the involvement of electrical connections in the generation of spontaneous network activity. In CA3 pyramidal cells, spikelets evoked by local stimulation of stratum oriens, were inhibited by carbenoxolone, but not by inhibitors of glutamatergic and GABAergic synaptic transmission, signifying the presence of electrical connectivity through axo-axonic gap junctions. Carbenoxolone also decreased the success rate of firing antidromic action potentials in response to stimulation, and changed the pattern of spontaneous action potential firing of CA3 pyramidal cells. Altogether, these data suggest that electrical coupling of CA3 pyramidal cells contribute to the generation of the early network events in neonatal hippocampus by modulating their firing pattern and synchronization. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Variations in gap junctional intercellular communication and connexin expression in fibroblasts derived from keloid and hypertrophic scars.

    PubMed

    Lu, Feng; Gao, JianHua; Ogawa, Rei; Hyakusoku, Hiko

    2007-03-01

    Expression of connexins and other constituent proteins of gap junctions along with gap junctional intercellular communication are involved in cellular development and differentiation processes. In addition, an increasing number of hereditary skin disorders appear to be linked to connexins. Therefore, in this report, the authors studied in vitro gap junctional intercellular communication function and connexin expression in fibroblasts derived from keloid and hypertrophic scar patients. Fibroblasts harvested from each of six keloid and hypertrophic scar patients were used for this study. Gap junctional intercellular communication function was investigated using the gap fluorescence recovery after photobleaching method, and expression of connexin proteins was studied using quantitative confocal microscopic analyses. Compared with normal skin, a decreased level of gap junctional intercellular communication was seen in fibroblasts derived from hypertrophic scar tissue, whereas an extremely low gap junctional intercellular communication level was detected in fibroblasts derived from keloid tissue. We also detected little connexin 43 (Cx43) protein localized in fibroblasts derived from keloids. Moreover, Cx43 protein levels were much lower in fibroblasts derived from hypertrophic scars than in those derived from normal skin. The authors' data suggest that the loss of gap junctional intercellular communication and connexin expression may affect intercellular recognition and thus break the proliferation and apoptosis balance in fibroblasts derived from keloid and hypertrophic scar tissue.

  16. pH-dependent modulation of connexin-based gap junctional uncouplers

    PubMed Central

    Skeberdis, Vytenis A; Rimkute, Lina; Skeberdyte, Aiste; Paulauskas, Nerijus; Bukauskas, Feliksas F

    2011-01-01

    Abstract Gap junction (GJ) channels formed from connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell–cell communication exhibiting high sensitivity to intracellular pH (pHi). We examined pHi-dependent modulation of junctional conductance (gj) of GJs formed of Cx26, mCx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols (LCCAs), arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH4Cl to pH ∼8 increased gj in cells expressing mCx30.2 and Cx45, yet did not affect gj of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 GJs. Unexpectedly, cells expressing Cx45, but not other Cxs, exhibited full coupling recovery after alkalization with NH4Cl under the continuous presence of LCCAs, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC50 for octanol was 0.1, 0.25 and 2.68 mm at pHi values of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide reduced the coupling-promoting effect of NH4Cl as well as the uncoupling effect of octanol. This suggests that LCCAs and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 GJ channel protein. PMID:21606109

  17. AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions

    PubMed Central

    Meyer, Arndt; Hilgen, Gerrit; Dorgau, Birthe; Sammler, Esther M.; Weiler, Reto; Monyer, Hannah; Dedek, Karin; Hormuzdi, Sheriar G.

    2014-01-01

    ABSTRACT Electrical synapses (gap junctions) rapidly transmit signals between neurons and are composed of connexins. In neurons, connexin36 (Cx36) is the most abundant isoform; however, the mechanisms underlying formation of Cx36-containing electrical synapses are unknown. We focus on homocellular and heterocellular gap junctions formed by an AII amacrine cell, a key interneuron found in all mammalian retinas. In mice lacking native Cx36 but expressing a variant tagged with enhanced green fluorescent protein at the C-terminus (KO-Cx36-EGFP), heterocellular gap junctions formed between AII cells and ON cone bipolar cells are fully functional, whereas homocellular gap junctions between two AII cells are not formed. A tracer injected into an AII amacrine cell spreads into ON cone bipolar cells but is excluded from other AII cells. Reconstruction of Cx36–EGFP clusters on an AII cell in the KO-Cx36-EGFP genotype confirmed that the number, but not average size, of the clusters is reduced – as expected for AII cells lacking a subset of electrical synapses. Our studies indicate that some neurons exhibit at least two discriminatory mechanisms for assembling Cx36. We suggest that employing different gap-junction-forming mechanisms could provide the means for a cell to regulate its gap junctions in a target-cell-specific manner, even if these junctions contain the same connexin. PMID:24463820

  18. Single-junction solar cells with the optimum band gap for terrestrial concentrator applications

    DOEpatents

    Wanlass, M.W.

    1994-12-27

    A single-junction solar cell is described having the ideal band gap for terrestrial concentrator applications. Computer modeling studies of single-junction solar cells have shown that the presence of absorption bands in the direct spectrum has the effect of ''pinning'' the optimum band gap for a wide range of operating conditions at a value of 1.14[+-]0.02 eV. Efficiencies exceeding 30% may be possible at high concentration ratios for devices with the ideal band gap. 7 figures.

  19. The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

    PubMed

    Rudolf, Mareike; Tetik, Nalan; Ramos-León, Félix; Flinner, Nadine; Ngo, Giang; Stevanovic, Mara; Burnat, Mireia; Pernil, Rafael; Flores, Enrique; Schleiff, Enrico

    2015-06-30

    mechanisms and components involved in bacteria are just emerging. Filamentous heterocyst-forming cyanobacteria behave as multicellular organisms and represent an excellent model to study prokaryotic cell-cell communication. A path for intercellular metabolite exchange appears to involve transfer through molecular structures termed septal junctions. They are reminiscent of metazoan gap junctions that directly link adjacent cells. In cyanobacteria, such structures need to traverse the peptidoglycan layers in the intercellular septa of the filament. Here we describe a factor involved in the formation of channels across the septal peptidoglycan layers, thus contributing to the multicellular behavior of these organisms. Copyright © 2015 Rudolf et al.

  20. Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis.

    PubMed

    Vaiyapuri, Sakthivel; Flora, Gagan D; Gibbins, Jonathan M

    2015-06-01

    Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischaemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a co-ordinated manner to prevent loss of blood and control wound repair. Previous report (1) indicates that the stability and functions of a thrombus are maintained through sustained, contact-dependent signalling between platelets. Given the role of gap junctions in the co-ordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

  1. Gap junction coupling is required for tumor cell migration through lymphatic endothelium.

    PubMed

    Karpinich, Natalie O; Caron, Kathleen M

    2015-05-01

    The lymphatic vasculature is a well-established conduit for metastasis, but the mechanisms by which tumor cells interact with lymphatic endothelial cells (LECs) to facilitate escape remain poorly understood. Elevated levels of the lymphangiogenic peptide adrenomedullin are found in many tumors, and we previously characterized that its expression is necessary for lymphatic vessel growth within both tumors and sentinel lymph nodes and for distant metastasis. This study used a tumor cell-LEC coculture system to identify a series of adrenomedullin-induced events that facilitated transendothelial migration of the tumor cells through a lymphatic monolayer. High levels of adrenomedullin expression enhanced adhesion of tumor cells to LECs, and further analysis revealed that adrenomedullin promoted gap junction coupling between LECs as evidenced by spread of Lucifer yellow dye. Adrenomedullin also enhanced heterocellular gap junction coupling as demonstrated by Calcein dye transfer from tumor cells into LECs. This connexin-mediated gap junction intercellular communication was necessary for tumor cells to undergo transendothelial migration because pharmacological blockade of this heterocellular communication prevented the ability of tumor cells to transmigrate through the lymphatic monolayer. In addition, treatment of LECs with adrenomedullin caused nuclear translocation of β-catenin, a component of endothelial cell junctions, causing an increase in transcription of the downstream target gene C-MYC. Importantly, blockade of gap junction intercellular communication prevented β-catenin nuclear translocation. Our findings indicate that maintenance of cell-cell communication is necessary to facilitate a cascade of events that lead to tumor cell migration through the lymphatic endothelium. © 2015 American Heart Association, Inc.

  2. Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway.

    PubMed

    Du, Zhong-Jun; Cui, Guan-Qun; Zhang, Juan; Liu, Xiao-Mei; Zhang, Zhi-Hu; Jia, Qiang; Ng, Jack C; Peng, Cheng; Bo, Cun-Xiang; Shao, Hua

    2017-01-01

    Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological

  3. ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCTED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS

    EPA Science Inventory

    ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS.
    OBJECTIVE: We have shown that functional gap junction communication as measured by Lucifer yellow dye transfer (DT) in Clone-9 rat liver epithelial cells, c...

  4. Role of gap junction intercellular communication in testicular leydig cell apoptosis induced by oxaliplatin via the mitochondrial pathway.

    PubMed

    Tong, Xuhui; Han, Xi; Yu, Binbin; Yu, Meiling; Jiang, Guojun; Ji, Jie; Dong, Shuying

    2015-01-01

    Platinum agents are widely used in the chemotherapy of testicular cancer. However, adverse reactions and resistance to such agents have limited their application in antineoplastic treatment. The aim of the present study was to determine the role of gap junction intercellular communication (GJIC) composed of Cx43 on oxaliplatin‑induced survival/apoptosis in mouse leydig normal and cancer cells using MTT, Annexin V/PI double staining assays and western blot analysis. The results showed that GJIC exerted opposite effects on the mouse leydig cancer (I-10) and normal (TM3) cell apoptosis induced by oxaliplatin. In leydig cancer cells, survival of cells exposed to oxaliplatin was substantially reduced when gap junctions formed as compared to no gap junctions. Pharmacological inhibition of gap junctions by oleamide and 18-α-glycyrrhetinic acid resulted in enhanced survival/decreased apoptosis while enhancement of gap junctions by retinoic acid led to decreased survival/increased apoptosis. These effects occurred only in high‑density cultures (gap junction formed), while the pharmacological modulations had no effects when there was no opportunity for gap junction formation. Notably, GJIC played an opposite (protective) role in normal leydig cells survival/apoptosis following exposure to oxaliplatin. Furthermore, this converse oxaliplatin‑inducing apoptosis exerted through the functional gap junction was correlated with the mitochondrial pathway‑related protein Bcl-2/Bax and caspase‑3/9. These results suggested that in testicular leydig normal/cancer cells, GJIC plays an opposite role in oxaliplatin‑induced apoptosis via the mitochondrial pathway.

  5. Gap junction turnover is achieved by the internalization of small endocytic double-membrane vesicles.

    PubMed

    Falk, Matthias M; Baker, Susan M; Gumpert, Anna M; Segretain, Dominique; Buckheit, Robert W

    2009-07-01

    Double-membrane-spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains ( approximately 0.05-0.5 mum in diameter) identified >30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles ( approximately 0.18-0.27 mum in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1-5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.

  6. Gap Junction Coupling and Calcium Waves in the Pancreatic Islet

    PubMed Central

    Benninger, Richard K. P.; Zhang, Min; Head, W. Steven; Satin, Leslie S.; Piston, David W.

    2008-01-01

    The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet. PMID:18805925

  7. Efficient encoding of motion is mediated by gap junctions in the fly visual system.

    PubMed

    Wang, Siwei; Borst, Alexander; Zaslavsky, Noga; Tishby, Naftali; Segev, Idan

    2017-12-01

    Understanding the computational implications of specific synaptic connectivity patterns is a fundamental goal in neuroscience. In particular, the computational role of ubiquitous electrical synapses operating via gap junctions remains elusive. In the fly visual system, the cells in the vertical-system network, which play a key role in visual processing, primarily connect to each other via axonal gap junctions. This network therefore provides a unique opportunity to explore the functional role of gap junctions in sensory information processing. Our information theoretical analysis of a realistic VS network model shows that within 10 ms following the onset of the visual input, the presence of axonal gap junctions enables the VS system to efficiently encode the axis of rotation, θ, of the fly's ego motion. This encoding efficiency, measured in bits, is near-optimal with respect to the physical limits of performance determined by the statistical structure of the visual input itself. The VS network is known to be connected to downstream pathways via a subset of triplets of the vertical system cells; we found that because of the axonal gap junctions, the efficiency of this subpopulation in encoding θ is superior to that of the whole vertical system network and is robust to a wide range of signal to noise ratios. We further demonstrate that this efficient encoding of motion by this subpopulation is necessary for the fly's visually guided behavior, such as banked turns in evasive maneuvers. Because gap junctions are formed among the axons of the vertical system cells, they only impact the system's readout, while maintaining the dendritic input intact, suggesting that the computational principles implemented by neural circuitries may be much richer than previously appreciated based on point neuron models. Our study provides new insights as to how specific network connectivity leads to efficient encoding of sensory stimuli.

  8. Does rat granulation tissue maturation involve gap junction communications?

    PubMed

    Au, Katherine; Ehrlich, H Paul

    2007-07-01

    Wound healing, a coordinated process, proceeds by sequential changes in cell differentiation and terminates with the deposition of a new connective tissue matrix, a scar. Initially, there is the migratory fibroblast, followed by the proliferative fibroblast, then the synthetic fibroblast, which transforms into the myofibroblast, and finally the apoptotic fibroblast. Gap junction intercellular communications are proposed to coordinate the stringent control of fibroblast phenotypic changes. Does added oleamide, a natural fatty acid that blocks gap junction intercellular communications, alter the phenotypic progression of wound fibroblasts? Pairs of polyvinyl alcohol sponges attached to Alzet pumps, which constantly pumped either oleamide or vehicle solvent, were implanted subcutaneously into three rats. On day 8, implants were harvested and evaluated histologically and biochemically. The capsule of oleamide-treated sponge contained closely packed fibroblasts with little connective tissue between them. The birefringence intensity of that connective tissue was reduced, indicating a reduced density of collagen fiber bundles. Myofibroblasts, identified immunohistologically by alpha-smooth muscle actin-stained stress fibers, were reduced in oleamide-treated implants. Western blot analysis showing less alpha-smooth muscle actin confirmed the reduced density of myofibroblasts. It appears that oleamide retards the progression of wound repair, where less connective tissue is deposited, the collagen is less organized, and the appearance of myofibroblasts is impaired. These findings support the hypothesis that gap junction intercellular communications between wound fibroblasts in granulation tissue play a role in the progression of repair and the maturation of granulation tissue into scar.

  9. Gap Junction Inhibition Prevents Drug-induced Liver Toxicity and Fulminant Hepatic Failure

    PubMed Central

    Patel, Suraj J; Milwid, Jack M; King, Kevin R; Bohr, Stefan; Iracheta, Arvin; Li, Matthew; Vitalo, Antonia; Parekkadan, Biju; Jindal, Rohit; Yarmush, Martin L

    2013-01-01

    Drug-induced liver injury (DILI) limits the development and utilization of numerous therapeutic compounds, and consequently presents major challenges to the pharmaceutical industry and clinical medicine1, 2. Acetaminophen (APAP) containing compounds are among the most frequently prescribed drugs, and also the most common cause of DILI3. Here we describe a pharmacological strategy that targets gap junction communication to prevent amplification of fulminant hepatic failure and APAP-induced hepatotoxicity. We report that connexin 32 (Cx32), a key hepatic gap junction protein, is an essential mediator of DILI by showing that mice deficient in Cx32 are protected against liver damage, acute inflammation, and death. We identified a small molecule inhibitor of Cx32 as a novel hepatoprotectant that achieves the same result in wildtype mice when coadministered with known hepatotoxic drugs. These findings demonstrate that gap junction inhibition is an effective therapy for limiting DILI, and suggest a novel pharmaceutical strategy to improve drug safety. PMID:22252509

  10. A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

    PubMed Central

    2011-01-01

    Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

  11. Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

    PubMed Central

    Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

    2014-01-01

    Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

  12. Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.

    PubMed

    Benninger, R K P; Head, W Steven; Zhang, Min; Satin, Leslie S; Piston, David W

    2011-11-15

    Cell-cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca(2+)](i)) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca(2+)](i) ceases when glucose is at a basal levels. We determine the roles of gap-junctions and other cell-cell communication pathways in the suppression of insulin secretion under basal conditions. Metabolic, electrical and insulin secretion levels were measured from islets lacking gap-junction coupling following deletion of connexion36 (Cx36(-/-)), and these results were compared to those obtained using fully isolated β-cells. K(ATP) loss-of-function islets provide a further experimental model to specifically study gap-junction mediated suppression of electrical activity. In isolated β-cells or Cx36(-/-) islets, elevations in [Ca(2+)](i) persisted in a subset of cells even at basal glucose. Isolated β-cells showed elevated insulin secretion at basal glucose; however, insulin secretion from Cx36(-/-) islets was minimally altered. [Ca(2+)](i) was further elevated under basal conditions, but insulin release still suppressed in K(ATP) loss-of-function islets. Forced elevation of cAMP led to PKA-mediated increases in insulin secretion from islets lacking gap-junctions, but not from islets expressing Cx36 gap junctions. We conclude there is a redundancy in how cell-cell communication in the islet suppresses insulin release. Gap junctions suppress cellular heterogeneity and spontaneous [Ca(2+)](i) signals, while other juxtacrine mechanisms, regulated by PKA and glucose, suppress more distal steps in exocytosis. Each mechanism is sufficiently robust to compensate for a loss of the other and still suppress basal insulin secretion.

  13. GAP JUNCTION COMMUNICATON IN A TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS

    EPA Science Inventory

    GAP JUNCTION COMMUNICTION IN TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS.

    OBJECTIVE: We previously showed that functional gap junction communication (GJC), as monitored by dye transfer (DT), could be enhanced in mouse C3H 10T112 cells and in mouse...

  14. Gap junction blockage promotes cadmium-induced apoptosis in BRL 3A derived from Buffalo rat liver cells.

    PubMed

    Hu, Di; Zou, Hui; Han, Tao; Xie, Junze; Dai, Nannan; Zhuo, Liling; Gu, Jianhong; Bian, Jianchun; Yuan, Yan; Liu, Xuezhong; Liu, Zongping

    2016-03-01

    Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca(2+) concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.

  15. Inhibiting connexin channels protects against cryopreservation-induced cell death in human blood vessels.

    PubMed

    Bol, M; Van Geyt, C; Baert, S; Decrock, E; Wang, N; De Bock, M; Gadicherla, A K; Randon, C; Evans, W H; Beele, H; Cornelissen, R; Leybaert, L

    2013-04-01

    Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability. Copyright © 2012 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

  16. Gap junctions and memory: an investigation using a single trial discrimination avoidance task for the neonate chick.

    PubMed

    Verwey, L J; Edwards, T M

    2010-02-01

    Gap junctions are important to how the brain functions but are relatively under-investigated with respect to their contribution towards behaviour. In the present study a single trial discrimination avoidance task was used to investigate the effect of the gap junction inhibitor 18-alpha-glycyrrhetinic acid (alphaGA) on retention. Past studies within our research group have implied a potential role for gap junctions during the short-term memory (STM) stage which decays by 15 min post-training. A retention function study comparing 10 microM alphaGA and vehicle given immediately post-training demonstrated a significant main effect for drug with retention loss at all times of test (10-180 min post-training). Given that the most common gap junction in the brain is that forming the astrocytic network it is reasonable to conclude that alphaGA was acting upon these. To confirm this finding and interpretation two additional investigations were undertaken using endothelin-1 (ET-1) and ET-1+tolbutamide. Importantly, a retention function study using 10nM ET-1 replicated the retention loss observed for alphaGA. In order to confirm that ET-1 was acting on astrocytic gap junctions the amnestic action of ET-1 was effectively challenged with increasing concentrations of tolbutamide. The present findings suggest that astrocytic gap junctions are important for memory processing. Copyright 2009 Elsevier Inc. All rights reserved.

  17. Gap junctions and hemichannels composed of connexins: potential therapeutic targets for neurodegenerative diseases

    PubMed Central

    Takeuchi, Hideyuki; Suzumura, Akio

    2014-01-01

    Microglia are macrophage-like resident immune cells that contribute to the maintenance of homeostasis in the central nervous system (CNS). Abnormal activation of microglia can cause damage in the CNS, and accumulation of activated microglia is a characteristic pathological observation in neurologic conditions such as trauma, stroke, inflammation, epilepsy, and neurodegenerative diseases. Activated microglia secrete high levels of glutamate, which damages CNS cells and has been implicated as a major cause of neurodegeneration in these conditions. Glutamate-receptor blockers and microglia inhibitors (e.g., minocycline) have been examined as therapeutic candidates for several neurodegenerative diseases; however, these compounds exerted little therapeutic benefit because they either perturbed physiological glutamate signals or suppressed the actions of protective microglia. The ideal therapeutic approach would hamper the deleterious roles of activated microglia without diminishing their protective effects. We recently found that abnormally activated microglia secrete glutamate via gap-junction hemichannels on the cell surface. Moreover, administration of gap-junction inhibitors significantly suppressed excessive microglial glutamate release and improved disease symptoms in animal models of neurologic conditions such as stroke, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. Recent evidence also suggests that neuronal and glial communication via gap junctions amplifies neuroinflammation and neurodegeneration. Elucidation of the precise pathologic roles of gap junctions and hemichannels may lead to a novel therapeutic strategies that can slow and halt the progression of neurodegenerative diseases. PMID:25228858

  18. Gap junctions on hippocampal mossy fiber axons demonstrated by thin-section electron microscopy and freeze–fracture replica immunogold labeling

    PubMed Central

    Hamzei-Sichani, Farid; Kamasawa, Naomi; Janssen, William G. M.; Yasumura, Thomas; Davidson, Kimberly G. V.; Hof, Patrick R.; Wearne, Susan L.; Stewart, Mark G.; Young, Steven R.; Whittington, Miles A.; Rash, John E.; Traub, Roger D.

    2007-01-01

    Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze–fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions (≈30–70 nm in diameter) were found between pairs of mossy fiber axons (≈100–200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction (≈100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy. PMID:17640909

  19. Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens.

    PubMed

    Biswas, Sondip K; Lee, Jai Eun; Brako, Lawrence; Jiang, Jean X; Lo, Woo-Kuen

    2010-11-09

    Ball-and-sockets and protrusions are specialized interlocking membrane domains between lens fibers of all species studied. Ball-and-sockets and protrusions are similar in their shape, size, and surface morphology, and are traditionally believed to play a key role in maintaining fiber-to-fiber stability. Here, we evaluate the hypothesis that ball-and-sockets and protrusions possess important structural and functional differences during fiber cell differentiation and maturation. Intact lenses of leghorn chickens (E7 days to P62 weeks old) and rhesus monkeys (1.5-20 years old) were studied with SEM, freeze-fracture TEM, freeze-fracture immunogold labeling (FRIL), and filipin cytochemistry for membrane cholesterol detection. SEM showed that ball-and-sockets were distributed along the long and short sides of hexagonal fiber cells, whereas protrusions were located along the cell corners, from superficial to deep cortical regions in both chicken and monkey lenses. Importantly, by freeze-fracture TEM, we discovered the selective association of gap junctions with all ball-and-sockets examined, but not with protrusions, in both species. In the embryonic chicken lens (E18), the abundant distribution of ball-and-socket gap junctions was regularly found in an approximate zone extending at least 300 μm deep from the equatorial surface of the superficial cortical fibers. Many ball-and-socket gap junctions often protruded deeply into neighboring cells. However, in the mature fibers of monkey lenses, several ball-and-sockets exhibited only partial occupancy of gap junctions with disorganized connexons, possibly due to degradation of gap junctions during fiber maturation and aging. FRIL analysis confirmed that both connexin46 (Cx46) and connexin50 (Cx50) antibodies specifically labeled ball-and-socket gap junctions, but not protrusions. Furthermore, filipin cytochemistry revealed that the ball-and-socket gap junctions contained different amounts of cholesterol (i.e., cholesterol

  20. Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens

    PubMed Central

    Biswas, Sondip K.; Lee, Jai Eun; Brako, Lawrence; Jiang, Jean X.

    2010-01-01

    Purpose Ball-and-sockets and protrusions are specialized interlocking membrane domains between lens fibers of all species studied. Ball-and-sockets and protrusions are similar in their shape, size, and surface morphology, and are traditionally believed to play a key role in maintaining fiber-to-fiber stability. Here, we evaluate the hypothesis that ball-and-sockets and protrusions possess important structural and functional differences during fiber cell differentiation and maturation. Methods Intact lenses of leghorn chickens (E7 days to P62 weeks old) and rhesus monkeys (1.5–20 years old) were studied with SEM, freeze-fracture TEM, freeze-fracture immunogold labeling (FRIL), and filipin cytochemistry for membrane cholesterol detection. Results SEM showed that ball-and-sockets were distributed along the long and short sides of hexagonal fiber cells, whereas protrusions were located along the cell corners, from superficial to deep cortical regions in both chicken and monkey lenses. Importantly, by freeze-fracture TEM, we discovered the selective association of gap junctions with all ball-and-sockets examined, but not with protrusions, in both species. In the embryonic chicken lens (E18), the abundant distribution of ball-and-socket gap junctions was regularly found in an approximate zone extending at least 300 μm deep from the equatorial surface of the superficial cortical fibers. Many ball-and-socket gap junctions often protruded deeply into neighboring cells. However, in the mature fibers of monkey lenses, several ball-and-sockets exhibited only partial occupancy of gap junctions with disorganized connexons, possibly due to degradation of gap junctions during fiber maturation and aging. FRIL analysis confirmed that both connexin46 (Cx46) and connexin50 (Cx50) antibodies specifically labeled ball-and-socket gap junctions, but not protrusions. Furthermore, filipin cytochemistry revealed that the ball-and-socket gap junctions contained different amounts of

  1. Inhibitors of connexin and pannexin channels as potential therapeutics

    PubMed Central

    Willebrords, Joost; Maes, Michaël; Crespo Yanguas, Sara; Vinken, Mathieu

    2018-01-01

    While gap junctions support the exchange of a number of molecules between neighboring cells, connexin hemichannels provide communication between the cytosol and the extracellular environment of an individual cell. The latter equally holds true for channels composed of pannexin proteins, which display an architecture reminiscent of connexin hemichannels. In physiological conditions, gap junctions are usually open, while connexin hemichannels and, to a lesser extent, pannexin channels are typically closed, yet they can be activated by a number of pathological triggers. Several agents are available to inhibit channels built up by connexin and pannexin proteins, including alcoholic substances, glycyrrhetinic acid, anesthetics and fatty acids. These compounds not always strictly distinguish between gap junctions, connexin hemichannels and pannexin channels, and may have effects on other targets as well. An exception lies with mimetic peptides, which reproduce specific amino acid sequences in connexin or pannexin primary protein structure. In this paper, a state-of-the-art overview is provided on inhibitors of cellular channels consisting of connexins and pannexins with specific focus on their mode-of-action and therapeutic potential. PMID:28720428

  2. Correlations among within-channel and between-channel auditory gap-detection thresholds in normal listeners.

    PubMed

    Phillips, Dennis P; Smith, Jennifer C

    2004-01-01

    We obtained data on within-channel and between-channel auditory temporal gap-detection acuity in the normal population. Ninety-five normal listeners were tested for gap-detection thresholds, for conditions in which the gap was bounded by spectrally identical, and by spectrally different, acoustic markers. Separate thresholds were obtained with the use of an adaptive tracking method, for gaps delimited by narrowband noise bursts centred on 1.0 kHz, noise bursts centred on 4.0 kHz, and for gaps bounded by a leading marker of 4.0 kHz noise and a trailing marker of 1.0 kHz noise. Gap thresholds were lowest for silent periods bounded by identical markers--'within-channel' stimuli. Gap thresholds were significantly longer for the between-channel stimulus--silent periods bounded by unidentical markers (p < 0.0001). Thresholds for the two within-channel tasks were highly correlated (R = 0.76). Thresholds for the between-channel stimulus were weakly correlated with thresholds for the within-channel stimuli (1.0 kHz, R = 0.39; and 4.0 kHz, R = 0.46). The relatively poor predictability of between-channel thresholds from the within-channel thresholds is new evidence on the separability of the mechanisms that mediate performance of the two tasks. The data confirm that the acuity difference for the tasks, which has previously been demonstrated in only small numbers of highly trained listeners, extends to a population of untrained listeners. The acuity of the between-channel mechanism may be relevant to the formation of voice-onset time-category boundaries in speech perception.

  3. Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus.

    PubMed

    Rash, J E; Olson, C O; Pouliot, W A; Davidson, K G V; Yasumura, T; Furman, C S; Royer, S; Kamasawa, N; Nagy, J I; Dudek, F E

    2007-10-26

    Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear

  4. Further evidence for the role of gap junctions in the delayed antiarrhythmic effect of cardiac pacing.

    PubMed

    Miskolczi, Gottfried; Gönczi, Márton; Kovács, Mária; Seprényi, György; Végh, Ágnes

    2015-07-01

    The objective of this study was to provide evidence that gap junctions are involved in the delayed antiarrhythmic effect of cardiac pacing. Twenty-four dogs were paced through the right ventricle (4 × 5 min, rate of 240 beats/min) 24 h prior to a 25 min occlusion of the left anterior descending coronary artery. Some of these paced dogs were infused with 50 (n = 7) or 100 μmol/L (n = 7) of the gap junction uncoupler carbenoxolone (CBX), prior to and during the occlusion. Ten sham-paced dogs, subjected only to occlusion, served as the controls. Cardiac pacing markedly reduced the number of ectopic beats and episodes of ventricular tachycardia (VT), as well the incidence of VT and ventricular fibrillation during occlusion. The changes in severity of ischaemia and tissue electrical resistance were also less marked compared with the unpaced controls. Pacing also preserved the permeability of gap junctions, the phosphorylation of connexin43, and the structural integrity of the intercalated discs. The closing of gap junctions with CBX prior to and during ischaemia markedly attenuated or even abolished these protective effects of pacing. Our results support the previous findings that gap junctions play a role in the delayed antiarrhythmic effect of cardiac pacing.

  5. Gap Junction Intercellular Communication: A Review of a Potential Platform to Modulate Craniofacial Tissue Engineering

    PubMed Central

    Rossello, Ricardo A.; Kohn, David H.

    2009-01-01

    Defects in craniofacial tissues, resulting from trauma, congenital abnormalities, oncologic resection or progressive deforming diseases, may result in aesthetic deformity, pain and reduced function. Restoring the structure, function and aesthetics of craniofacial tissues represents a substantial clinical problem in need of new solutions. More biologically-interactive biomaterials could potentially improve the treatment of craniofacial defects, and an understanding of developmental processes may help identify strategies and materials that can be used in tissue engineering. One such strategy that can potentially advance tissue engineering is cell–cell communication. Gap junction intercellular communication is the most direct way of achieving such signaling. Gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering. Following a review of gap junctions in bone cells, the ability to enhance cell–cell communication and osteogenic differentiation via control of gap junctions is discussed, as is the potential utility of this approach in craniofacial tissue engineering. PMID:18481782

  6. Subsets of ATP-sensitive potassium channel (KATP) inhibitors increase gap junctional intercellular communication in metastatic cancer cell lines independent of SUR expression

    USDA-ARS?s Scientific Manuscript database

    Gap junctional intercellular communication (GJIC) is a process whereby cells share molecules and nutrients with each other by physical contact through cell membrane pores. In tumor cells, GJIC is often altered, suggesting that this process may be important in the context of cancer. Certain ion chan...

  7. Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip.

    PubMed

    Tellez-Gabriel, Marta; Charrier, Céline; Brounais-Le Royer, Bénédicte; Mullard, Mathilde; Brown, Hannah K; Verrecchia, Franck; Heymann, Dominique

    2017-03-01

    Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours - such as bone tumours - is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a dielectrophoresis (DEP)-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics. Copyright © 2017 Elsevier GmbH. All rights reserved.

  8. The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice.

    PubMed

    Zhou, Cheng-Jie; Wu, Sha-Na; Shen, Jiang-Peng; Wang, Dong-Hui; Kong, Xiang-Wei; Lu, Angeleem; Li, Yan-Jiao; Zhou, Hong-Xia; Zhao, Yue-Fang; Liang, Cheng-Guang

    2016-01-01

    Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo- or in vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

  9. Methamphetamine compromises gap junctional communication in astrocytes and neurons

    PubMed Central

    Castellano, Paul; Nwagbo, Chisom; Martinez, Luis R.; Eugenin, Eliseo A.

    2016-01-01

    Methamphetamine (meth) is a central nervous system (CNS) stimulant that results in psychological and physical dependency. The long-term effects of meth within the CNS include neuronal plasticity changes, blood–brain barrier compromise, inflammation, electrical dysfunction, neuronal/glial toxicity, and an increased risk to infectious diseases including HIV. Most of the reported meth effects in the CNS are related to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters, especially dopamine, norepinephrine, and epinephrine. However, little is known about the effects of meth on connexin (Cx) containing channels, such as gap junctions (GJ) and hemichannels (HC). We examined the effects of meth on Cx expression, function, and its role in NeuroAIDS. We found that meth altered Cx expression and localization, decreased GJ communication between neurons and astrocytes, and induced the opening of Cx43/Cx36 HC. Furthermore, we found that these changes in GJ and HC induced by meth treatment were mediated by activation of dopamine receptors, suggesting that dysregulation of dopamine signaling induced by meth is essential for GJ and HC compromise. Meth-induced changes in GJ and HC contributed to amplified CNS toxicity by dysregulating glutamate metabolism and increasing the susceptibility of neurons and astrocytes to bystander apoptosis induced by HIV. Together, our results indicate that connexin containing channels, GJ and HC, are essential in the pathogenesis of meth and increase the sensitivity of the CNS to HIV CNS disease. PMID:26953131

  10. ABUNDANCE AND ULTRASTRUCTURAL DIVERSITY OF NEURONAL GAP JUNCTIONS IN THE OFF AND ON SUBLAMINAE OF THE INNER PLEXIFORM LAYER OF RAT AND MOUSE RETINA

    PubMed Central

    KAMASAWA, N.; FURMAN, C. S.; DAVIDSON, K. G. V.; SAMPSON, J. A.; MAGNIE, A. R.; GEBHARDT, B. R.; KAMASAWA, M.; YASUMURA, T.; ZUMBRUNNEN, J. R.; PICKARD, G. E.; NAGY, J. I.; RASH, J. E.

    2007-01-01

    Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under “baseline” conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline “plaques” (71% and 3%), plus unusual “string” (14%), “ribbon” (7%) and “reticular” (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter

  11. Abundance and ultrastructural diversity of neuronal gap junctions in the OFF and ON sublaminae of the inner plexiform layer of rat and mouse retina.

    PubMed

    Kamasawa, N; Furman, C S; Davidson, K G V; Sampson, J A; Magnie, A R; Gebhardt, B R; Kamasawa, M; Yasumura, T; Zumbrunnen, J R; Pickard, G E; Nagy, J I; Rash, J E

    2006-11-03

    Neuronal gap junctions are abundant in both outer and inner plexiform layers of the mammalian retina. In the inner plexiform layer (IPL), ultrastructurally-identified gap junctions were reported primarily in the functionally-defined and anatomically-distinct ON sublamina, with few reported in the OFF sublamina. We used freeze-fracture replica immunogold labeling and confocal microscopy to quantitatively analyze the morphologies and distributions of neuronal gap junctions in the IPL of adult rat and mouse retina. Under "baseline" conditions (photopic illumination/general anesthesia), 649 neuronal gap junctions immunogold-labeled for connexin36 were identified in rat IPL, of which 375 were photomapped to OFF vs. ON sublaminae. In contrast to previous reports, the volume-density of gap junctions was equally abundant in both sublaminae. Five distinctive morphologies of gap junctions were identified: conventional crystalline and non-crystalline "plaques" (71% and 3%), plus unusual "string" (14%), "ribbon" (7%) and "reticular" (2%) forms. Plaque and reticular gap junctions were distributed throughout the IPL. However, string and ribbon gap junctions were restricted to the OFF sublamina, where they represented 48% of gap junctions in that layer. In string and ribbon junctions, curvilinear strands of connexons were dispersed over 5 to 20 times the area of conventional plaques having equal numbers of connexons. To define morphologies of gap junctions under different light-adaptation conditions, we examined an additional 1150 gap junctions from rats and mice prepared after 30 min of photopic, mesopic and scotopic illumination, with and without general anesthesia. Under these conditions, string and ribbon gap junctions remained abundant in the OFF sublamina and absent in the ON sublamina. Abundant gap junctions in the OFF sublamina of these two rodents with rod-dominant retinas revealed previously-undescribed but extensive pathways for inter-neuronal communication; and the

  12. Promotion of lens epithelial-fiber differentiation by the C-terminus of connexin 45.6 a role independent of gap junction communication.

    PubMed

    Banks, Eric A; Yu, X Sean; Shi, Qian; Jiang, Jean X

    2007-10-15

    We previously reported that, among the three connexins expressed in chick lens, overexpression of connexin (Cx) 45.6, not Cx43 or Cx56, stimulates lens cell differentiation; however, the underlying mechanism responsible for this effect is unclear. Here, we took advantage of naturally occurring loss-of-gap-junction function mutations of Cx50 (ortholog of chick Cx45.6) and generated the corresponding site mutants in Cx45.6: Cx45.6(D47A) and Cx45.6(P88S). In contrast to wild-type Cx45.6, the mutants failed to form functional gap junctions, and Cx45.6(P88S) and, to a lesser degree, Cx45.6(D47A) functioned in a dominant-negative manner. Interestingly, overexpression of both mutants incapable of forming gap junctions significantly increased epithelial-fiber differentiation to a level comparable to that of wild-type Cx45.6. To map the functional domain of Cx45.6, we generated a C-terminus chimera as well as deletion mutants. Overexpression of Cx56(*)45.6C, the mutant in which the C-terminus of Cx56 was replaced with that of Cx45.6, had a stimulatory effect on lens cell differentiation similar to that of Cx45.6. However, cells overexpressing Cx45.6(*)56C, the mutant in which C-terminus of Cx45.6 was replaced with that of Cx56, and Cx45.6(-C), in which the C-terminus was deleted, failed to promote differentiation. Taken together, we conclude that the expression of Cx45.6, but not Cx45.6-dependent gap junction channels, is involved in lens epithelial-fiber cell differentiation, and the C-terminal domain of Cx45.6 plays a predominant role in mediating this process.

  13. Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1.

    PubMed

    Chu, Miensheng; Novak, Stefanie Mares; Cover, Cathleen; Wang, Anne A; Chinyere, Ikeotunye Royal; Juneman, Elizabeth B; Zarnescu, Daniela C; Wong, Pak Kin; Gregorio, Carol C

    2018-02-06

    Gap junction remodeling is well established as a consistent feature of human heart disease involving spontaneous ventricular arrhythmia. The mechanisms responsible for gap junction remodeling that include alterations in the distribution of, and protein expression within, gap junctions are still debated. Studies reveal that multiple transcriptional and posttranscriptional regulatory pathways are triggered in response to cardiac disease, such as those involving RNA-binding proteins. The expression levels of FXR1 (fragile X mental retardation autosomal homolog 1), an RNA-binding protein, are critical to maintain proper cardiac muscle function; however, the connection between FXR1 and disease is not clear. To identify the mechanisms regulating gap junction remodeling in cardiac disease, we sought to identify the functional properties of FXR1 expression, direct targets of FXR1 in human left ventricle dilated cardiomyopathy (DCM) biopsy samples and mouse models of DCM through BioID proximity assay and RNA immunoprecipitation, how FXR1 regulates its targets through RNA stability and luciferase assays, and functional consequences of altering the levels of this important RNA-binding protein through the analysis of cardiac-specific FXR1 knockout mice and mice injected with 3xMyc-FXR1 adeno-associated virus. FXR1 expression is significantly increased in tissue samples from human and mouse models of DCM via Western blot analysis. FXR1 associates with intercalated discs, and integral gap junction proteins Cx43 (connexin 43), Cx45 (connexin 45), and ZO-1 (zonula occludens-1) were identified as novel mRNA targets of FXR1 by using a BioID proximity assay and RNA immunoprecipitation. Our findings show that FXR1 is a multifunctional protein involved in translational regulation and stabilization of its mRNA targets in heart muscle. In addition, introduction of 3xMyc-FXR1 via adeno-associated virus into mice leads to the redistribution of gap junctions and promotes ventricular

  14. The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

    PubMed

    Wang, Hong-Xing; Gillio-Meina, Carolina; Chen, Shuli; Gong, Xiang-Qun; Li, Tony Y; Bai, Donglin; Kidder, Gerald M

    2013-08-01

    WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

  15. Entrainment, retention, and transport of freely swimming fish in junction gaps between commercial barges operating on the Illinois Waterway

    USGS Publications Warehouse

    Davis, Jeremiah J.; Jackson, P. Ryan; Engel, Frank; LeRoy, Jessica Z.; Neeley, Rebecca N.; Finney, Samuel T.; Murphy, Elizabeth A.

    2016-01-01

    Large Electric Dispersal Barriers were constructed in the Chicago Sanitary and Ship Canal (CSSC) to prevent the transfer of invasive fish species between the Mississippi River Basin and the Great Lakes Basin while simultaneously allowing the passage of commercial barge traffic. We investigated the potential for entrainment, retention, and transport of freely swimming fish within large gaps (> 50 m3) created at junction points between barges. Modified mark and capture trials were employed to assess fish entrainment, retention, and transport by barge tows. A multi-beam sonar system enabled estimation of fish abundance within barge junction gaps. Barges were also instrumented with acoustic Doppler velocity meters to map the velocity distribution in the water surrounding the barge and in the gap formed at the junction of two barges. Results indicate that the water inside the gap can move upstream with a barge tow at speeds near the barge tow travel speed. Water within 1 m to the side of the barge junction gaps was observed to move upstream with the barge tow. Observed transverse and vertical water velocities suggest pathways by which fish may potentially be entrained into barge junction gaps. Results of mark and capture trials provide direct evidence that small fish can become entrained by barges, retained within junction gaps, and transported over distances of at least 15.5 km. Fish entrained within the barge junction gap were retained in that space as the barge tow transited through locks and the Electric Dispersal Barriers, which would be expected to impede fish movement upstream.

  16. Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma

    PubMed Central

    Kumar, Sandeep; Ramakrishnan, Hariharasubramanian; Roy, Kaushambi; Viswanathan, Suresh; Bloomfield, Stewart A.

    2017-01-01

    The progressive death of retinal ganglion cells and resulting visual deficits are hallmarks of glaucoma, but the underlying mechanisms remain unclear. In many neurodegenerative diseases, cell death induced by primary insult is followed by a wave of secondary loss. Gap junctions (GJs), intercellular channels composed of subunit connexins, can play a major role in secondary cell death by forming conduits through which toxic molecules from dying cells pass to and injure coupled neighbors. Here we have shown that pharmacological blockade of GJs or genetic ablation of connexin 36 (Cx36) subunits, which are highly expressed by retinal neurons, markedly reduced loss of neurons and optic nerve axons in a mouse model of glaucoma. Further, functional parameters that are negatively affected in glaucoma, including the electroretinogram, visual evoked potential, visual spatial acuity, and contrast sensitivity, were maintained at control levels when Cx36 was ablated. Neuronal GJs may thus represent potential therapeutic targets to prevent the progressive neurodegeneration and visual impairment associated with glaucoma. PMID:28604388

  17. Nonlinear Gap Junctions Enable Long-Distance Propagation of Pulsating Calcium Waves in Astrocyte Networks

    PubMed Central

    Goldberg, Mati; De Pittà, Maurizio; Volman, Vladislav; Berry, Hugues; Ben-Jacob, Eshel

    2010-01-01

    A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca2+ waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP3) through gap junctions that locally trigger Ca2+ pulses via IP3-dependent Ca2+-induced Ca2+ release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca2+ waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca2+ wave propagation in astrocytes. Our model yields two major predictions. First, we show that long-distance regenerative signaling requires nonlinear coupling in the gap junctions. Second, we show that even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca2+ dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca2+ waves across astrocytes and the precise conditions under which glial cells may participate in information processing in the brain. PMID:20865153

  18. GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

    PubMed

    Pinet-Charvet, Caroline; Geller, Sarah; Desroziers, Elodie; Ottogalli, Monique; Lomet, Didier; Georgelin, Christine; Tillet, Yves; Franceschini, Isabelle; Vaudin, Pascal; Duittoz, Anne

    2016-01-01

    Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

  19. Lipopolysaccharide effects on the proliferation of NRK52E cells via alternations in gap-junction function.

    PubMed

    Hei, Ziqing; Zhang, Ailan; Wei, Jing; Gan, Xiaoliang; Wang, Yanling; Luo, Gangjian; Li, Xiaoyun

    2012-07-01

    Gap junctions regulate proper kidney function by facilitating intercellular communication, vascular conduction, and tubular purinergic signaling. However, no clear relationship has been described between gap-junction function and acute kidney injury induced by the endotoxin lipopolysaccharide (LPS). Normal rat kidney epithelial cells (NRK52E cells) were seeded at high and low densities to promote or impede gap-junction formation, respectively, and establish distinctive levels of intercellular communication in culture. Cells were then challenged with LPS at various concentrations (10-1,000 ng/mL). LPS-induced formation and function of gap junctions were assessed by measuring changes in cell proliferation and colony-forming rates, fluorescent dye transmission to adjacent cells, expression levels of connexin43, and repositioning of confluent cells in response to the gap junction inhibitor oleamide or agonist retinoic acid. The cell proliferation rate and colony-forming rate of high- and low-density NRK52E cells were decreased upon LPS challenge, in a dose-dependent manner. The colony-forming rate of confluent high-density cells was significantly lower than that of low-density cells. Oleamide treatment raised the LPS-induced colony-forming rate of high-density cells, whereas retinoic acid decreased the rate. Neither oleamide nor retinoic acid significantly affected the LPS-induced colony-forming rate of low-density cells. Fluorescence transmission of high-density cells was reduced by LPS challenge, in a dose-dependent manner, but inclusion of retinoic acid increased the LPS-induced transmission of fluorescence. LPS challenge of either high- or low-density NRK52E cells resulted in down-regulated connexin43 expression. Gap-junction function plays an important role in concentration-dependent cytotoxic effect of LPS on normal rat kidney cells in vitro.

  20. Bioavailability and efficacy of a gap junction enhancer (PQ7) in a mouse mammary tumor model.

    PubMed

    Shishido, Stephanie N; Prasain, Keshar; Beck, Amanda; Nguyen, Thi D T; Hua, Duy H; Nguyen, Thu Annelise

    2013-01-01

    The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.

  1. Short-term depression of gap junctional coupling in reticular thalamic neurons of absence epileptic rats.

    PubMed

    Kohmann, Denise; Lüttjohann, Annika; Seidenbecher, Thomas; Coulon, Philippe; Pape, Hans-Christian

    2016-10-01

    Gap junctional electrical coupling between neurons of the reticular thalamic nucleus (RTN) is critical for hypersynchrony in the thalamo-cortical network. This study investigates the role of electrical coupling in pathological rhythmogenesis in RTN neurons in a rat model of absence epilepsy. Rhythmic activation resulted in a Ca(2+) -dependent short-term depression (STD) of electrical coupling between pairs of RTN neurons in epileptic rats, but not in RTN of a non-epileptic control strain. Pharmacological blockade of gap junctions in RTN in vivo induced a depression of seizure activity. The STD of electrical coupling represents a mechanism of Ca(2+) homeostasis in RTN aimed to counteract excessive synchronization. Neurons in the reticular thalamic nucleus (RTN) are coupled by electrical synapses, which play a major role in regulating synchronous activity. This study investigates electrical coupling in RTN neurons from a rat model of childhood absence epilepsy, genetic absence epilepsy rats from Strasbourg (GAERS), compared with a non-epileptic control (NEC) strain, to assess the impact on pathophysiological rhythmogenesis. Whole-cell recordings were obtained from pairs of RTN neurons of GAERS and NEC in vitro. Coupling was determined by injection of hyperpolarizing current steps in one cell and monitoring evoked voltage responses in both activated and coupled cell. The coupling coefficient (cc) was compared under resting condition, during pharmacological interventions and repeated activation using a series of current injections. The effect of gap junctional coupling on seizure expression was investigated by application of gap junctional blockers into RTN of GAERS in vivo. At resting conditions, cc did not differ between GAERS and NEC. During repeated activation, cc declined in GAERS but not in NEC. This depression in cc was restored within 25 s and was prevented by intracellular presence of BAPTA in the activated but not in the coupled cell. Local application of

  2. FREQUENCY-DEPENDENT CHANGES IN GAP JUNCTION FUNCTION IN PRIMARY HEPATOCYTES

    EPA Science Inventory

    FREQUENCY-DEPENDENT CHANGES IN GAP JUNCTION FUNCTION IN PRIMARY HEPATOCYTES. X. Wang1 *, D.E. Housel *, J. Page2, C.F. Blackmanl. 1 National Health and Environmental Effects Research Laboratory, USEPA, Research Triangle Park, North Carolina 27711 USA, 2Oakland, California USA
    ...

  3. Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

    PubMed

    Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

    2014-01-17

    Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

  4. A unified framework for spiking and gap-junction interactions in distributed neuronal network simulations.

    PubMed

    Hahne, Jan; Helias, Moritz; Kunkel, Susanne; Igarashi, Jun; Bolten, Matthias; Frommer, Andreas; Diesmann, Markus

    2015-01-01

    Contemporary simulators for networks of point and few-compartment model neurons come with a plethora of ready-to-use neuron and synapse models and support complex network topologies. Recent technological advancements have broadened the spectrum of application further to the efficient simulation of brain-scale networks on supercomputers. In distributed network simulations the amount of spike data that accrues per millisecond and process is typically low, such that a common optimization strategy is to communicate spikes at relatively long intervals, where the upper limit is given by the shortest synaptic transmission delay in the network. This approach is well-suited for simulations that employ only chemical synapses but it has so far impeded the incorporation of gap-junction models, which require instantaneous neuronal interactions. Here, we present a numerical algorithm based on a waveform-relaxation technique which allows for network simulations with gap junctions in a way that is compatible with the delayed communication strategy. Using a reference implementation in the NEST simulator, we demonstrate that the algorithm and the required data structures can be smoothly integrated with existing code such that they complement the infrastructure for spiking connections. To show that the unified framework for gap-junction and spiking interactions achieves high performance and delivers high accuracy in the presence of gap junctions, we present benchmarks for workstations, clusters, and supercomputers. Finally, we discuss limitations of the novel technology.

  5. Chloral hydrate decreases gap junction communications in rat liver epithelial cells

    EPA Science Inventory

    Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involvedareunknown.Chloralhydrate(CH), a by-productofchlorinedisinfection ofwater,is carcinogenic in mice,...

  6. Gap junction networks can generate both ripple-like and fast ripple-like oscillations

    PubMed Central

    Simon, Anna; Traub, Roger D.; Vladimirov, Nikita; Jenkins, Alistair; Nicholson, Claire; Whittaker, Roger G.; Schofield, Ian; Clowry, Gavin J.; Cunningham, Mark O.; Whittington, Miles A.

    2014-01-01

    Fast ripples (FRs) are network oscillations, defined variously as having frequencies of > 150 to > 250 Hz, with a controversial mechanism. FRs appear to indicate a propensity of cortical tissue to originate seizures. Here, we demonstrate field oscillations, at up to 400 Hz, in spontaneously epileptic human cortical tissue in vitro, and present a network model that could explain FRs themselves, and their relation to ‘ordinary’ (slower) ripples. We performed network simulations with model pyramidal neurons, having axons electrically coupled. Ripples (< 250 Hz) were favored when conduction of action potentials, axon to axon, was reliable. Whereas ripple population activity was periodic, firing of individual axons varied in relative phase. A switch from ripples to FRs took place when an ectopic spike occurred in a cell coupled to another cell, itself multiply coupled to others. Propagation could then start in one direction only, a condition suitable for re-entry. The resulting oscillations were > 250 Hz, were sustained or interrupted, and had little jitter in the firing of individual axons. The form of model FR was similar to spontaneously occurring FRs in excised human epileptic tissue. In vitro, FRs were suppressed by a gap junction blocker. Our data suggest that a given network can produce ripples, FRs, or both, via gap junctions, and that FRs are favored by clusters of axonal gap junctions. If axonal gap junctions indeed occur in epileptic tissue, and are mediated by connexin 26 (recently shown to mediate coupling between immature neocortical pyramidal cells), then this prediction is testable. PMID:24118191

  7. Gap junction networks can generate both ripple-like and fast ripple-like oscillations.

    PubMed

    Simon, Anna; Traub, Roger D; Vladimirov, Nikita; Jenkins, Alistair; Nicholson, Claire; Whittaker, Roger G; Schofield, Ian; Clowry, Gavin J; Cunningham, Mark O; Whittington, Miles A

    2014-01-01

    Fast ripples (FRs) are network oscillations, defined variously as having frequencies of > 150 to > 250 Hz, with a controversial mechanism. FRs appear to indicate a propensity of cortical tissue to originate seizures. Here, we demonstrate field oscillations, at up to 400 Hz, in spontaneously epileptic human cortical tissue in vitro, and present a network model that could explain FRs themselves, and their relation to 'ordinary' (slower) ripples. We performed network simulations with model pyramidal neurons, having axons electrically coupled. Ripples (< 250 Hz) were favored when conduction of action potentials, axon to axon, was reliable. Whereas ripple population activity was periodic, firing of individual axons varied in relative phase. A switch from ripples to FRs took place when an ectopic spike occurred in a cell coupled to another cell, itself multiply coupled to others. Propagation could then start in one direction only, a condition suitable for re-entry. The resulting oscillations were > 250 Hz, were sustained or interrupted, and had little jitter in the firing of individual axons. The form of model FR was similar to spontaneously occurring FRs in excised human epileptic tissue. In vitro, FRs were suppressed by a gap junction blocker. Our data suggest that a given network can produce ripples, FRs, or both, via gap junctions, and that FRs are favored by clusters of axonal gap junctions. If axonal gap junctions indeed occur in epileptic tissue, and are mediated by connexin 26 (recently shown to mediate coupling between immature neocortical pyramidal cells), then this prediction is testable. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  8. Gap junctions are essential for murine primordial follicle assembly immediately before birth.

    PubMed

    Teng, Zhen; Wang, Chao; Wang, Yijing; Huang, Kun; Xiang, Xi; Niu, Wanbao; Feng, Lizhao; Zhao, Lihua; Yan, Hao; Zhang, Hua

    2016-02-01

    The reserve of primordial follicles determines the reproductive ability of the female mammal over its reproductive life. The primordial follicle is composed of two types of cells: oocytes and surrounding pre-granulosa cells. However, the underlying mechanism regulating primordial follicle assembly is largely undefined. In this study, we found that gap junction communication (GJC) established between the ovarian cells in the perinatal mouse ovary may be involved in the process. First, gap junction structures between the oocyte and surrounding pre-granulosa cells appear at about 19.0 dpc (days post coitum). As many as 12 gap junction-related genes are upregulated at birth, implying that a complex communication may exist between ovarian cells, because specifically silencing the genes of individual gap junction proteins, such as Gja1, Gja4 or both, has no influence on primordial follicle assembly. On the other hand, non-specific blockers of GJC, such as carbenoxolone (CBX) and 18α-glycyrrhetinic acid (AGA), significantly inhibit mouse primordial follicle assembly. We proved that the temporal window for establishment of GJC in the fetal ovary is from 19.5 dpc to 1 dpp (days postpartum). In addition, the expression of ovarian somatic cell (OSC)-specific genes, such as Notch2, Foxl2 and Irx3, was negatively affected by GJC blockers, whereas oocyte-related genes, such as Ybx2, Nobox and Sohlh1, were hardly affected, implying that the establishment of GJC during this period may be more important to OSCs than to oocytes. In summary, our results indicated that GJC involves in the mouse primordial follicle assembly process at a specific temporal window that needs Notch signaling cross-talking. © 2016 Society for Reproduction and Fertility.

  9. Genetic variants related to gap junctions and hormone secretion influence conception rates in cows.

    PubMed

    Sugimoto, Mayumi; Sasaki, Shinji; Gotoh, Yusaku; Nakamura, Yuuki; Aoyagi, Yoshito; Kawahara, Takayoshi; Sugimoto, Yoshikazu

    2013-11-26

    The recent decline in fertility is a serious problem in the dairy industry. To overcome this problem, we performed a genome-wide association study using 384 Holsteins and identified four loci associated with conception rates. Two of them contained gap junction-related genes: PKP2 and CTTNBP2NL. Further analysis confirmed that PKP2 increased connexin 43, a gap junction protein, whereas CTTNBP2NL dephosphorylated connexin 43. Knockdown of PKP2 or overexpression of CTTNBP2NL inhibited embryo implantation in mice. The other two loci contained neuroendocrine-related genes: SETD6 and CACNB2. Additional experiments indicated that SETD6 is involved in the transcriptional regulation of gonadotropin-releasing hormone, whereas CACNB2 controlled the secretion of follicle-stimulating hormone in cattle. The total allele substitution effect of these genes on conception rate was 3.5%. Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry.

  10. Propofol depresses cisplatin cytotoxicity via the inhibition of gap junctions.

    PubMed

    Zhang, Yuan; Wang, Xiyan; Wang, Qin; Ge, Hui; Tao, Liang

    2016-06-01

    The general anesthetic, propofol, affects chemotherapeutic activity, however, the mechanism underlying its effects remains to be fully elucidated. Our previous study showed that tramadol and flurbiprofen depressed the cytotoxicity of cisplatin via the inhibition of gap junction (GJ) intercellular communication (GJIC) in connexin (Cx)32 HeLa cells. The present study investigated whether the effects of propofol on the cytotoxicity of cisplatin were mediated by GJ in U87 glioma cells and Cx26‑transfected HeLa cells. Standard colony formation assay was used to determine the cytotoxicity of cisplatin. Parachute dye coupling assay was used to measure GJ function, and western blot analysis was used to determine the expression levels of Cx32. The results revealed that exposure of the U87 glioma cells and the Cx26-transfected HeLa cells to cisplatin for 1 h reduced clonogenic survival in low density cultures (without GJs) and high density cultures (with GJs). However, the toxic effect was higher in the high density culture. In addition, pretreatment of the cells with propofol significantly reduced cisplatin‑induced cytotoxicity, but only in the presence of functional GJs. Furthermore, propofol significantly inhibited dye coupling through junctional channels, and a long duration of exposure of the cells to propofol downregulated the expression levels of Cx43 and Cx26. These results demonstrated that the inhibition of GJIC by propofol affected the therapeutic efficacy of chemotherapeutic drugs. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting chemotherapeutic efficiency.

  11. Gap junction-dependent homolog avoidance in the developing CNS.

    PubMed

    Baker, Michael W; Yazdani, Neema; Macagno, Eduardo R

    2013-10-16

    Oppositely directed projections of some homologous neurons in the developing CNS of the medicinal leech (Hirudo verbana), such as the AP cells, undergo a form of contact-dependent homolog avoidance. Embryonic APs extend axons within the connective nerve toward adjacent ganglia, in which they meet and form gap junctions (GJs) with the oppositely directed axons of their segmental homologs, stop growing, and are later permanently retracted (Wolszon et al., 1994a,b). However, early deletion of an AP neuron leads to resumed growth and permanent maintenance of the projections of neighboring APs. Here we test the hypothesis that a GJ-based signaling mechanism is responsible for this instance of homolog avoidance. We demonstrate that selective knockdown of GJ gene Hve-inx1 expression in single embryonic APs, by expressing a short-hairpin interfering RNA, leads to continued growth of the projections of the cell toward, into, and beyond adjacent ganglia. Moreover, the projections of the APs in adjacent ganglia also resume growth, mimicking their responses to cell deletion. Continued growth was also observed when two different INX1 mutant transgenes that abolish dye coupling between APs were expressed. These include a mutant transgene that effectively downregulates all GJ plaques that include the INX1 protein and a closed channel INX1 mutant that retains the adhesive cellular binding characteristic of INX1 GJs but not the open channel pore function. Our results add GJ intercellular communication to the list of molecular signaling mechanisms that can act as mediators of growth-inhibiting cell-cell interactions that define the topography of neuronal arbors.

  12. Chemical Synaptic and Gap Junctional Interactions Between Principal Neurons: Partners in Epileptogenesis

    PubMed Central

    Traub, Roger D.; Cunningham, Mark O.; Whittington, Miles A.

    2010-01-01

    Field potential signals, corresponding to electrographic seizures in cortical structures, often contain two components, which sometimes appear to be separable and other times to be superimposed. The first component consists of low-amplitude very fast oscillations (VFO, > 70–80 Hz); the second component consists of larger amplitude transients, lasting tens to hundreds of ms, and variously called population spikes, EEG spikes, or bursts – terms chosen in part because of the cellular correlates of the field events. To first approximation, the two components arise because of distinctive types of cellular interactions: gap junctions for VFO (a model of which is reviewed in the following), and recurrent synaptic excitation and/or inhibition for the transients. With in vitro studies of epileptic human neocortical tissue, it is possible to elicit VFO alone, or VFO superimposed on a large transient, but not a large transient without the VFO. If such observations prove to be general, they would imply that gap junction-mediated interactions are the primary factor in epileptogenesis. It appears to be the case then, that in the setting of seizure initiation (but not necessarily under physiological conditions), the gain of gap junction-mediated circuits can actually be larger than the gain in excitatory synaptic circuits. PMID:21168305

  13. Chemical synaptic and gap junctional interactions between principal neurons: partners in epileptogenesis.

    PubMed

    Traub, Roger D; Cunningham, Mark O; Whittington, Miles A

    2011-08-01

    Field potential signals, corresponding to electrographic seizures in cortical structures, often contain two components, which sometimes appear to be separable and other times to be superimposed. The first component consists of low-amplitude very fast oscillations (VFO, >70-80 Hz); the second component consists of larger amplitude transients, lasting tens to hundreds of ms, and variously called population spikes, EEG spikes, or bursts--terms chosen in part because of the cellular correlates of the field events. To first approximation, the two components arise because of distinctive types of cellular interactions: gap junctions for VFO (a model of which is reviewed in the following), and recurrent synaptic excitation and/or inhibition for the transients. With in vitro studies of epileptic human neocortical tissue, it is possible to elicit VFO alone, or VFO superimposed on a large transient, but not a large transient without the VFO. If such observations prove to be general, they would imply that gap junction-mediated interactions are the primary factor in epileptogenesis. It appears to be the case then, that in the setting of seizure initiation (but not necessarily under physiological conditions), the gain of gap junction-mediated circuits can actually be larger than the gain in excitatory synaptic circuits. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. A unified framework for spiking and gap-junction interactions in distributed neuronal network simulations

    PubMed Central

    Hahne, Jan; Helias, Moritz; Kunkel, Susanne; Igarashi, Jun; Bolten, Matthias; Frommer, Andreas; Diesmann, Markus

    2015-01-01

    Contemporary simulators for networks of point and few-compartment model neurons come with a plethora of ready-to-use neuron and synapse models and support complex network topologies. Recent technological advancements have broadened the spectrum of application further to the efficient simulation of brain-scale networks on supercomputers. In distributed network simulations the amount of spike data that accrues per millisecond and process is typically low, such that a common optimization strategy is to communicate spikes at relatively long intervals, where the upper limit is given by the shortest synaptic transmission delay in the network. This approach is well-suited for simulations that employ only chemical synapses but it has so far impeded the incorporation of gap-junction models, which require instantaneous neuronal interactions. Here, we present a numerical algorithm based on a waveform-relaxation technique which allows for network simulations with gap junctions in a way that is compatible with the delayed communication strategy. Using a reference implementation in the NEST simulator, we demonstrate that the algorithm and the required data structures can be smoothly integrated with existing code such that they complement the infrastructure for spiking connections. To show that the unified framework for gap-junction and spiking interactions achieves high performance and delivers high accuracy in the presence of gap junctions, we present benchmarks for workstations, clusters, and supercomputers. Finally, we discuss limitations of the novel technology. PMID:26441628

  15. Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

    NASA Technical Reports Server (NTRS)

    Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

    2003-01-01

    Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

  16. Regulation of blood-testis barrier dynamics by desmosome, gap junction, hemidesmosome and polarity proteins

    PubMed Central

    Wong, Elissa WP; Lie, Pearl PY; Li, Michelle WM; Mruk, Dolores D; Yan, Helen HN; Mok, Ka-Wai; Mannu, Jayakanthan; Mathur, Premendu P; Lui, Wing-yee; Lee, Will M; Bonanomi, Michele; Silvestrini, Bruno

    2011-01-01

    The blood-testis barrier (BTB) is a unique ultrastructure in the mammalian testis. Unlike other blood-tissue barriers, such as the blood-brain barrier and the blood-ocular (or blood-retina) barrier which formed by tight junctions (TJ) between endothelial cells of the microvessels, the BTB is constituted by coexisting TJ, basal ectoplasmic specialization (basal ES), desmosomes and gap junctions between adjacent Sertoli cells near the basement membrane of the seminiferous tubule. The BTB also divides the seminiferous epithelium into the apical (or adluminal) and basal compartments so that meiosis I and II and post-meiotic germ cell development can all take place in a specialized microenvironment in the apical compartment behind the BTB. While the unusual anatomical features of the BTB have been known for decades, the physiological function of the coexisting junctions, in particular the desmosome and gap junction, that constitute the BTB was unknown until recently. Based on recently published findings, we critically evaluate the role of the desmosome and gap junction that serve as a signaling platform to coordinate the “opening” and “closing” of the TJ-permeability barrier conferred by TJ and basal ES during the seminiferous epithelial cycle of spermatogenesis. This is made possible by polarity proteins working in concert with nonreceptor protein tyrosine kinases, such as focal adhesion kinase (FAK) and c-Src, at the site to regulate endosome-mediated protein trafficking events (e.g., endocytosis, transcytosis, recycling or protein degradation). These events not only serve to destabilize the existing “old” BTB above preleptotene spermatocytes in transit in “clones” at the BTB, but also contribute to the assembly of “new” BTB below the transiting spermatocytes. Furthermore, hemidesmosomes at the Sertoli cell-basement membrane interface also contribute to the BTB restructuring events at stage VIII of the epithelial cycle. Additionally, the findings

  17. Gap Junction Intercellular Communication Mediates Ammonia-Induced Neurotoxicity.

    PubMed

    Bobermin, Larissa Daniele; Arús, Bernardo Assein; Leite, Marina Concli; Souza, Diogo Onofre; Gonçalves, Carlos-Alberto; Quincozes-Santos, André

    2016-02-01

    Astrocytes are important brain targets of ammonia, a neurotoxin implicated in the development of hepatic encephalopathy. During hyperammonemia, the pivotal role of astrocytes in brain function and homeostasis is impaired. These cells are abundantly interconnected by gap junctions (GJ), which are intercellular channels that allow the exchange of signaling molecules and metabolites. This communication may also increase cellular vulnerability during injuries, while GJ uncoupling could limit the extension of a lesion. Therefore, the current study was performed to investigate whether astrocyte coupling through GJ contributes to ammonia-induced cytotoxicity. We found that carbenoxolone (CBX), an effective GJ blocker, prevented the following effects induced by ammonia in astrocyte primary cultures: (1) decrease in cell viability and membrane integrity; (2) increase in reactive oxygen species production; (3) decrease in GSH intracellular levels; (4) GS activity; (5) pro-inflammatory cytokine release. On the other hand, CBX had no effect on C6 astroglial cells, which are poorly coupled via GJ. To our knowledge, this study provides the first evidence that GJ play a role in ammonia-induced cytotoxicity. Although more studies in vivo are required to confirm our hypothesis, our data suggest that GJ communication between astrocytes may transmit damage signals and excitotoxic components from unhealthy to normal cells, thereby contributing to the propagation of the neurotoxicity of ammonia.

  18. Intrinsic islet heterogeneity and gap junction coupling determine spatiotemporal Ca²⁺ wave dynamics.

    PubMed

    Benninger, Richard K P; Hutchens, Troy; Head, W Steven; McCaughey, Michael J; Zhang, Min; Le Marchand, Sylvain J; Satin, Leslie S; Piston, David W

    2014-12-02

    Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca(2+)]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca(2+)]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca(2+)]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca(2+)]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  19. Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ*

    PubMed Central

    Farnsworth, Nikki L.; Walter, Rachelle L.; Hemmati, Alireza; Westacott, Matthew J.; Benninger, Richard K. P.

    2016-01-01

    Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. PMID:26668311

  20. Low Level Pro-inflammatory Cytokines Decrease Connexin36 Gap Junction Coupling in Mouse and Human Islets through Nitric Oxide-mediated Protein Kinase Cδ.

    PubMed

    Farnsworth, Nikki L; Walter, Rachelle L; Hemmati, Alireza; Westacott, Matthew J; Benninger, Richard K P

    2016-02-12

    Pro-inflammatory cytokines contribute to the decline in islet function during the development of diabetes. Cytokines can disrupt insulin secretion and calcium dynamics; however, the mechanisms underlying this are poorly understood. Connexin36 gap junctions coordinate glucose-induced calcium oscillations and pulsatile insulin secretion across the islet. Loss of gap junction coupling disrupts these dynamics, similar to that observed during the development of diabetes. This study investigates the mechanisms by which pro-inflammatory cytokines mediate gap junction coupling. Specifically, as cytokine-induced NO can activate PKCδ, we aimed to understand the role of PKCδ in modulating cytokine-induced changes in gap junction coupling. Isolated mouse and human islets were treated with varying levels of a cytokine mixture containing TNF-α, IL-1β, and IFN-γ. Islet dysfunction was measured by insulin secretion, calcium dynamics, and gap junction coupling. Modulators of PKCδ and NO were applied to determine their respective roles in modulating gap junction coupling. High levels of cytokines caused cell death and decreased insulin secretion. Low levels of cytokine treatment disrupted calcium dynamics and decreased gap junction coupling, in the absence of disruptions to insulin secretion. Decreases in gap junction coupling were dependent on NO-regulated PKCδ, and altered membrane organization of connexin36. This study defines several mechanisms underlying the disruption to gap junction coupling under conditions associated with the development of diabetes. These mechanisms will allow for greater understanding of islet dysfunction and suggest ways to ameliorate this dysfunction during the development of diabetes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Genetic variants related to gap junctions and hormone secretion influence conception rates in cows

    PubMed Central

    Sugimoto, Mayumi; Sasaki, Shinji; Gotoh, Yusaku; Nakamura, Yuuki; Aoyagi, Yoshito; Kawahara, Takayoshi; Sugimoto, Yoshikazu

    2013-01-01

    The recent decline in fertility is a serious problem in the dairy industry. To overcome this problem, we performed a genome-wide association study using 384 Holsteins and identified four loci associated with conception rates. Two of them contained gap junction-related genes: PKP2 and CTTNBP2NL. Further analysis confirmed that PKP2 increased connexin 43, a gap junction protein, whereas CTTNBP2NL dephosphorylated connexin 43. Knockdown of PKP2 or overexpression of CTTNBP2NL inhibited embryo implantation in mice. The other two loci contained neuroendocrine-related genes: SETD6 and CACNB2. Additional experiments indicated that SETD6 is involved in the transcriptional regulation of gonadotropin-releasing hormone, whereas CACNB2 controlled the secretion of follicle-stimulating hormone in cattle. The total allele substitution effect of these genes on conception rate was 3.5%. Our findings reveal important roles for gap junction communication and the neuroendocrine system in conception and suggest unique selection methods to improve reproductive performance in the livestock industry. PMID:24218568

  2. The effect of nerve section on the incidence and distribution of gap junctions in the odontoblast layer of the cat.

    PubMed

    Holland, G R

    1987-08-01

    Gap junctions are numerous in the odontoblast layer of the dental pulp and may link sensory axons to odontoblasts. If these junctions do link axons and odontoblasts, they, together with the axons, should disappear after cutting the pulpal nerves centrally. Under general anesthesia the inferior alveolar nerve on one side of two young adult cats was sectioned. Under general anesthesia the animals were perfused with fixative 56 hours later and the coronal dental pulp prepared for electron microscopy. Ultrathin sections were examined from the level of the pulpal cornu and levels approximately one, two, and three mm below this. The incidence of cell processes and gap junctions was measured at different distances from the pulp predentin junction, and operated and control sides compared. The odontoblast layer at the level of the cornu differed from elsewhere in having, on the control side, a greater density of cell processes and gap junctions and in having clearly recognizable axons approaching to within 5 to 10 micron of the predentin. The only statistically significant changes after nerve section occurred in this layer and consisted of a decline in the incidence of cell processes and of gap junctions that link one cell process to another. There was no significant difference between the operated and control sides in the number of gap junctions linking cell processes to recognizable cell bodies. The odontoblast layer in the pulpal cornu contained substantial numbers of unsheathed axons, many presumably en route to the dentin. These axons may participate in gap junctions that link them to other cell processes, possibly even other axons.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. PI3K/Akt signaling is involved in the disruption of gap junctional communication caused by v-Src and TNF-α.

    PubMed

    Ito, Satoko; Hyodo, Toshinori; Hasegawa, Hitoki; Yuan, Hong; Hamaguchi, Michinari; Senga, Takeshi

    2010-09-17

    Gap junctional communication, which is mediated by the connexin protein family, is essential for the maintenance of normal tissue function and homeostasis. Loss of intercellular communication results in a failure to coordinately regulate cellular functions, and it can facilitate tumorigenesis. Expression of oncogenes and stimulation with cytokines has been shown to suppress intercellular communication; however, the exact mechanism by which intercellular communication is disrupted by these factors remains uncertain. In this report, we show that Akt is essential for the disruption of gap junctional communication in v-Src-transformed cells. In addition, inhibition of Akt restores gap junctional communication after it is suppressed by TNF-α signaling. Furthermore, we demonstrate that the expression of a constitutively active form of Akt1, but not of Akt2 or Akt3, is sufficient to suppress gap junctional communication. Our results clearly define Akt1 as one of the critical regulators of gap junctional communication. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Gap-Junctional communication between developing Drosophila muscles is essential for their normal development.

    PubMed

    Todman, M G; Baines, R A; Stebbings, L A; Davies, J A; Bacon, J P

    1999-01-01

    Recent experiments have demonstrated that a family of proteins, known as the innexins, are structural components of invertebrate gap junctions. The shaking-B (shak-B) locus of Drosophila encodes two members of this emerging family, Shak-B(lethal) and Shak-B(neural). This study focuses on the role of Shak-B gap junctions in the development of embryonic and larval muscle. During embryogenesis, shak-B transcripts are expressed in a subset of the somatic muscles; expression is strong in ventral oblique muscles (VO4-6) but only weak in ventral longitudinals (VL3 and 4). Carboxyfluorescein injected into VO4 of wild-type early stage 16 embryos spreads, via gap junctions, to label adjacent muscles, including VL3 and 4. In shak-B2 embryos (in which the shak-B(neural) function is disrupted), dye injected into VO4 fails to spread into other muscles. In the first instar larva, when dye coupling between muscles is no longer present, another effect of the shak-B2 mutation is revealed by whole-cell voltage clamp. In a calcium-free saline, only two voltage-activated potassium currents are present in wild-type muscles; a fast IA and a slow IK current. In shak-B2 larvae, these two currents are significantly reduced in magnitude in VO4 and 5, but remain normal in VL3. Expression of shak-B(neural) in a shak-B2 background fully rescues both dye coupling in embryonic muscle and whole-cell currents in first instar VO4 and 5. Our observations show that Shak-B(neural) is one of a set of embryonic gap-junction proteins, and that it is required for the normal temporal development of potassium currents in some larval muscles.

  5. GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

    PubMed Central

    Mäkinen, Meeri Eeva-Liisa; Ylä-Outinen, Laura; Narkilahti, Susanna

    2018-01-01

    The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling. PMID:29559893

  6. Electronic and Thermal Effects in the Insulator-Metal Phase Transition in VO2 Nano-Gap Junctions

    DTIC Science & Technology

    2014-11-27

    VO2 , air, or SiO2, the 2 -0.50 -0.25 0.00 0.25 0.50 0 2 4 6 V Gap V App V o lt ag e (V ) time (ms) t p V I→M V M→I 100 400 700 1000 0.0 2.5 5.0 7.5...Electronic and thermal effects in the insulator-metal phase transition in VO2 nano-gap junctions Arash Joushaghani,1 Junho Jeong,1 Suzanne Paradis,2...Canada (Dated: 27 November 2014) By controlling the thermal transport of VO2 nano-gap junctions using device geometry, contact material, and applied

  7. A decay of gap junctions associated with ganglion cell differentiation during retinal regeneration of the adult newt.

    PubMed

    Oi, Hanako; Chiba, Chikafumi; Saito, Takehiko

    2003-12-01

    Changes in the gap junctional coupling and maturation of voltage-activated Na(+) currents during regeneration of newt retinas were examined by whole-cell patch-clamping in slice preparations. Progenitor cells in regenerating retinas did not exhibit Na(+) currents but showed prominent electrical and tracer couplings. Cells identified by LY-fills were typically slender. Na(+) currents were detected in premature ganglion cells with round somata in the 'intermediate-II' regenerating retina. No electrical and tracer couplings were observed between these cells. Mature ganglion cells did not exhibit electrical coupling, but showed tracer coupling. On average, the maximum Na(+) current amplitude recorded from premature ganglion cells was roughly 2.5-fold smaller than that of mature ganglion cells. In addition, the activation threshold of the Na(+) current was nearly 11 mV more positive than that of mature cells. We provide morphological and physiological evidence showing that loss of gap junctions between progenitor cells is associated with ganglion cell differentiation during retinal regeneration and that new gap junctions are recreated between mature ganglion cells. Also we provide evidence suggesting that the loss of gap junctions correlates with the appearance of voltage-activated Na(+) currents in ganglion cells.

  8. Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques.

    PubMed

    Stout, Randy F; Snapp, Erik Lee; Spray, David C

    2015-09-25

    Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. The alpha2-adrenoreceptor agonist dexmedetomidine protects against lipopolysaccharide-induced apoptosis via inhibition of gap junctions in lung fibroblasts.

    PubMed

    Zhang, Yuan; Tan, Xiaoming; Xue, Lianfang

    2018-01-01

    The α2-adrenoceptor inducer dexmedetomidine protects against acute lung injury (ALI), but the mechanism of this effect is largely unknown. The present study investigated the effect of dexmedetomidine on apoptosis induced by lipopolysaccharide (LPS) and the relationship between this effect and gap junction intercellular communication in human lung fibroblast cell line. Flow cytometry was used to detect apoptosis induced by LPS. Parachute dye coupling assay was used to measure gap junction function, and western blot analysis was used to determine the expression levels of connexin43 (Cx43). The results revealed that exposure of human lung fibroblast cell line to LPS for 24 h increased the apoptosis, and pretreatment of dexmedetomidine and 18α-GA significantly reduced LPS-induced apoptosis. Dexmedetomidine exposure for 1 h inhibited gap junction function mainly via a decrease in Cx43 protein levels in human lung fibroblast cell line. These results demonstrated that the inhibition of gap junction intercellular communication by dexmedetomidine affected the LPS-induced apoptosis through inhibition of gap junction function by reducing Cx43 protein levels. The present study provides evidence of a novel mechanism underlying the effects of analgesics in counteracting ALI. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Calcium dynamics in cardiac excitatory and non-excitatory cells and the role of gap junction.

    PubMed

    Das, Phonindra Nath; Mehrotra, Parul; Mishra, Aseem; Bairagi, Nandadulal; Chatterjee, Samrat

    2017-07-01

    Calcium ions aid in the generation of action potential in myocytes and are responsible for the excitation-contraction coupling of heart. The heart muscle has specialized patches of cells, called excitatory cells (EC) such as the Sino-atrial node cells capable of auto-generation of action potential and cells which receive signals from the excitatory cells, called non-excitatory cells (NEC) such as cells of the ventricular and auricular walls. In order to understand cardiac calcium homeostasis, it is, therefore, important to study the calcium dynamics taking into account both types of cardiac cells. Here we have developed a model to capture the calcium dynamics in excitatory and non-excitatory cells taking into consideration the gap junction mediated calcium ion transfer from excitatory cell to non-excitatory cell. Our study revealed that the gap junctional coupling between excitatory and non-excitatory cells plays important role in the calcium dynamics. It is observed that any reduction in the functioning of gap junction may result in abnormal calcium oscillations in NEC, even when the calcium dynamics is normal in EC cell. Sensitivity of gap junction is observed to be independent of the pacing rate and hence a careful monitoring is required to maintain normal cardiomyocyte condition. It also highlights that sarcoplasmic reticulum may not be always able to control the amount of cytoplasmic calcium under the condition of calcium overload. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy.

    PubMed

    Lemcke, Heiko; Voronina, Natalia; Steinhoff, Gustav; David, Robert

    2017-06-19

    Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

  12. Homeostatic Plasticity Mediated by Rod-Cone Gap Junction Coupling in Retinal Degenerative Dystrophic RCS Rats

    PubMed Central

    Hou, Baoke; Fu, Yan; Weng, Chuanhuang; Liu, Weiping; Zhao, Congjian; Yin, Zheng Qin

    2017-01-01

    Rod-cone gap junctions open at night to allow rod signals to pass to cones and activate the cone-bipolar pathway. This enhances the ability to detect large, dim objects at night. This electrical synaptic switch is governed by the circadian clock and represents a novel form of homeostatic plasticity that regulates retinal excitability according to network activity. We used tracer labeling and ERG recording in the retinae of control and retinal degenerative dystrophic RCS rats. We found that in the control animals, rod-cone gap junction coupling was regulated by the circadian clock via the modulation of the phosphorylation of the melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT). However, in dystrophic RCS rats, AANAT was constitutively phosphorylated, causing rod-cone gap junctions to remain open. A further b/a-wave ratio analysis revealed that dystrophic RCS rats had stronger synaptic strength between photoreceptors and bipolar cells, possibly because rod-cone gap junctions remained open. This was despite the fact that a decrease was observed in the amplitude of both a- and b-waves as a result of the progressive loss of rods during early degenerative stages. These results suggest that electric synaptic strength is increased during the day to allow cone signals to pass to the remaining rods and to be propagated to rod bipolar cells, thereby partially compensating for the weak visual input caused by the loss of rods. PMID:28473754

  13. Intercellular communication in sensory ganglia by purinergic receptors and gap junctions: implications for chronic pain.

    PubMed

    Hanani, Menachem

    2012-12-03

    Peripheral injury can cause abnormal activity in sensory neurons, which is a major factor in chronic pain. Recent work has shown that injury induces major changes not only in sensory neurons but also in the main type of glial cells in sensory ganglia-satellite glial cells (SGCs), and that interactions between sensory neurons and SGCs contribute to neuronal activity in pain models. The main functional changes observed in SGCs after injury are an increased gap junction-mediated coupling among these cells, and augmented sensitivity to ATP. There is evidence that the augmented gap junctions contribute to neuronal hyperexcitability in pain models, but the mechanism underlying this effect is not known. The changes in SGCs described above have been found following a wide range of injuries (both axotomy and inflammation) in somatic, orofacial and visceral regions, and therefore appear to be a general feature in chronic pain. We have found that in cultures of sensory ganglia calcium signals can spread from an SGC to neighboring cells by calcium waves, which are mediated by gap junctions and ATP acting on purinergic P2 receptors. A model is proposed to explain how augmented gap junctions and greater sensitivity to ATP can combine to produce enhanced calcium waves, which can lead to neuronal excitation. Thus this simple scheme can account for several major changes in sensory ganglia that are common to a great variety of pain models. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Aberrant activity in retinal degeneration impairs central visual processing and relies on Cx36-containing gap junctions.

    PubMed

    Ivanova, Elena; Yee, Christopher W; Baldoni, Robert; Sagdullaev, Botir T

    2016-09-01

    In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

    PubMed

    Ma, Ke-Tao; Li, Xin-Zhi; Li, Li; Jiang, Xue-Wei; Chen, Xin-Yan; Liu, Wei-Dong; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang

    2014-02-01

    To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

  16. Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

    PubMed

    Hichri, Echrak; Abriel, Hugues; Kucera, Jan P

    2018-02-15

    It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na + ) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na + channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na + channels, we show that restricting the extracellular space modulates the Na + current, as predicted by corresponding simulations accounting for ephaptic effects. In a high-resolution model of the intercalated disc, clusters of Na + channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na + channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na + current (I Na ) are scarce. Furthermore, Na + channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Na v 1.5 channels, we examined how restricting the extracellular space modulates I Na elicited by an activation protocol. In parallel, we developed a high-resolution computer model of the intercalated disc to investigate how the distribution of Na + channels influences ephaptic interactions. Approaching the HEK cells to a non-conducting obstacle always increased peak I Na at step potentials near the threshold of I Na activation

  17. Terbinafine inhibits gap junctional intercellular communication.

    PubMed

    Lee, Ju Yeun; Yoon, Sei Mee; Choi, Eun Ju; Lee, Jinu

    2016-09-15

    Terbinafine is an antifungal agent that selectively inhibits fungal sterol synthesis by blocking squalene epoxidase. We evaluated the effect of terbinafine on gap junctional intercellular communication (GJIC). Fluorescence recovery after photobleaching (FRAP) and I-YFP GJIC assays revealed that terbinafine inhibits GJIC in a reversible and dose-dependent manner in FRT-Cx43 and LN215 cells. Treatment with terbinafine did not affect Cx43 phosphorylation status or intracellular Ca(2+) concentration, well-known action mechanisms of various GJIC blockers. While a structurally related chemical, naftifine, attenuated GJIC, epigallocatechin gallate, another potent squalene epoxidase inhibitor with a different structure, did not. These results suggest that terbinafine inhibits GJIC with a so far unknown mechanism of action. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The distribution and functional properties of Pelizaeus-Merzbacher-like disease-linked Cx47 mutations on Cx47/Cx47 homotypic and Cx47/Cx43 heterotypic gap junctions.

    PubMed

    Kim, Mi Seong; Gloor, Gregory B; Bai, Donglin

    2013-06-01

    GJs (gap junctions) allow direct intercellular communication, and consist of Cxs (connexins). In the mammalian central nervous system, oligodendrocytes express Cx47, Cx32 and Cx29, whereas astrocytes express Cx43, Cx30 and Cx26. Homotypic Cx47/Cx47 GJs couple oligodendrocytes, and heterotypic Cx47/Cx43 channels are the primary GJs at oligodendrocyte/astrocyte junctions. Interestingly, autosomal recessive mutations in the gene GJC2 encoding Cx47 have been linked to a central hypomyelinating disease termed PMLD (Pelizaeus-Merzbacher-like disease). The aim of the present study was to determine the cellular distribution and functional properties of PMLD-associated Cx47 mutants (I46M, G149S, G236R, G236S, M286T and T398I). Expressing GFP (green fluorescent protein)-tagged mutant versions of Cx47 in gap-junction-deficient model cells revealed that these mutants were detected at the cell-cell interface similar to that observed for wild-type Cx47. Furthermore, four of the six mutants showed no electrical coupling in both Cx47/Cx47 and Cx47/Cx43 GJ channels. These results suggest that most of the PMLD-linked Cx47 mutants disrupt Cx47/Cx47 and Cx47/Cx43 GJ function in the glial network, which may play a role in leading to PMLD symptoms.

  19. INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

    EPA Science Inventory


    Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
    Communication

    Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
    Laboratory, Office of Research and Development, US EPA, Research Triang...

  20. Role of gap junctional coupling in astrocytic networks in the determination of global ischaemia-induced oxidative stress and hippocampal damage.

    PubMed

    Perez Velazquez, Jose L; Kokarovtseva, Larisa; Sarbaziha, Raheleh; Jeyapalan, Zina; Leshchenko, Yevgen

    2006-01-01

    While there is evidence that gap junctions play important roles in the determination of cell injuries, there is not much known about mechanisms by which gap junctional communication may exert these functions. Using a global model of transient ischaemia in rats, we found that pretreatment with the gap junctional blockers carbenoxolone, 18alpha-glycyrrhetinic acid and endothelin, applied via cannulae implanted into the hippocampus in one hemisphere, resulted in decreased numbers of TUNEL-positive neurons, as compared with the contralateral hippocampus that received saline injection. Post-treatment with carbenoxolone for up to 30 min after the stroke injury still resulted in decreased cell death, but post-treatment at 90 min after the ischaemic insult did not result in differences in cell death. However, quinine, an inhibitor of Cx36-mediated gap junctional coupling, did not result in appreciable neuroprotection. Searching for a possible mechanism for the observed protective effects, possible actions of the gap junctional blockers in the electrical activity of the hippocampus during the ischaemic insult were assessed using intracerebral recordings, with no differences observed between the saline-injected and the contralateral drug-injected hippocampus. However, a significant reduction in lipid peroxides, a measure of free radical formation, in the hippocampus treated with carbenoxolone, revealed that the actions of gap junctional coupling during injuries may be causally related to oxidative stress. These observations suggest that coupling in glial networks may be functionally important in determining neuronal vulnerability to oxidative injuries.

  1. Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury.

    PubMed

    Calì, Bianca; Ceolin, Stefano; Ceriani, Federico; Bortolozzi, Mario; Agnellini, Andrielly H R; Zorzi, Veronica; Predonzani, Andrea; Bronte, Vincenzo; Molon, Barbara; Mammano, Fabio

    2015-04-30

    Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding "bystander" cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca(2+)-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy.

  2. Critical role of gap junction communication, calcium and nitric oxide signaling in bystander responses to focal photodynamic injury

    PubMed Central

    Calì, Bianca; Ceolin, Stefano; Ceriani, Federico; Bortolozzi, Mario; Agnellini, Andrielly H.R.; Zorzi, Veronica; Predonzani, Andrea; Bronte, Vincenzo

    2015-01-01

    Ionizing and nonionizing radiation affect not only directly targeted cells but also surrounding “bystander” cells. The underlying mechanisms and therapeutic role of bystander responses remain incompletely defined. Here we show that photosentizer activation in a single cell triggers apoptosis in bystander cancer cells, which are electrically coupled by gap junction channels and support the propagation of a Ca2+ wave initiated in the irradiated cell. The latter also acts as source of nitric oxide (NO) that diffuses to bystander cells, in which NO levels are further increased by a mechanism compatible with Ca2+-dependent enzymatic production. We detected similar signals in tumors grown in dorsal skinfold chambers applied to live mice. Pharmacological blockade of connexin channels significantly reduced the extent of apoptosis in bystander cells, consistent with a critical role played by intercellular communication, Ca2+ and NO in the bystander effects triggered by photodynamic therapy. PMID:25868859

  3. Cx43 in mesenchymal stem cells promotes angiogenesis of the infarcted heart independent of gap junctions.

    PubMed

    Wang, De-Guo; Zhang, Feng-Xiang; Chen, Ming-Long; Zhu, Hong-Jun; Yang, Bing; Cao, Ke-Jiang

    2014-04-01

    Mesenchymal stem cells (MSCs) with elevated levels of connexin 43 (Cx43) have been shown to exhibit improved protection for ischemic hearts. However, it remains unclear whether Cx43 is involved in the paracrine actions of angiogenesis, the major mechanism of cell therapy. In the present study, an in vitro model with deprivation of oxygen and a murine myocardial infarction model with permanent ligation of the left anterior‑descending (LAD) coronary artery were used to determine whether gap junctions in MSCs promote angiogenesis. It was observed that MSCs that overexpressed Cx43 (MSCs‑Cx43), improve the cardiac function of infarcted myocardium as compared with control MSCs (MSCs‑vector) and MSCs with Cx43 knocked down by small interfering RNA (MSCs‑siCx43), accompanied with a reduction of infarct size and an increase in the vascular density and maturity. Increased levels of representative angiogenic factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] were produced by MSCs‑Cx43 compared with MSCs‑siCx43 in vivo and in vitro. However, neither Cx43 formed gap junction specific inhibitor (Cx43 mimetic peptide) or gap junction opener (antiarrhythmic peptide) affected the production of VEGF and bFGF in MSCs under hypoxic stress. These data support the hypothesis that Cx43 in MSCs promotes angiogenesis in the infarcted heart, independent of gap junction formation.

  4. Connexin composition in apposed gap junction hemiplaques revealed by matched double-replica freeze-fracture replica immunogold labeling.

    PubMed

    Rash, John E; Kamasawa, Naomi; Davidson, Kimberly G V; Yasumura, Thomas; Pereda, Alberto E; Nagy, James I

    2012-06-01

    Despite the combination of light-microscopic immunocytochemistry, histochemical mRNA detection techniques and protein reporter systems, progress in identifying the protein composition of neuronal versus glial gap junctions, determination of the differential localization of their constituent connexin proteins in two apposing membranes and understanding human neurological diseases caused by connexin mutations has been problematic due to ambiguities introduced in the cellular and subcellular assignment of connexins. Misassignments occurred primarily because membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques. Currently, only serial thin-section transmission electron microscopy and freeze-fracture replica immunogold labeling have sufficient resolution to assign connexin proteins to either or both sides of gap junction plaques. However, freeze-fracture replica immunogold labeling has been limited because conventional freeze fracturing allows retrieval of only one of the two membrane fracture faces within a gap junction, making it difficult to identify connexin coupling partners in hemiplaques removed by fracturing. We now summarize progress in ascertaining the connexin composition of two coupled hemiplaques using matched double-replicas that are labeled simultaneously for multiple connexins. This approach allows unambiguous identification of connexins and determination of the membrane "sidedness" and the identities of connexin coupling partners in homotypic and heterotypic gap junctions of vertebrate neurons.

  5. Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

    PubMed

    Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

    2011-01-01

    Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  6. Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats.

    PubMed

    Patrone, Luis G A; Bícego, Kênia Cardoso; Hartzler, Lynn K; Putnam, Robert W; Gargaglioni, Luciane H

    2014-01-01

    The locus coeruleus (LC) plays an important role in central chemoreception. In young rats (P9 or younger), 85% of LC neurons increase firing rate in response to hypercapnia vs. only about 45% of neurons from rats P10 or older. Carbenoxolone (CARB - gap junction blocker) does not affect the % of LC neurons responding in young rats but it decreases the % responding by half in older animals. We evaluated the participation of gap junctions in the CO2 ventilatory response in unanesthetized adult rats by bilaterally microinjecting CARB (300μM, 1mM or 3mM/100nL), glycyrrhizic acid (GZA, CARB analog, 3mM) or vehicle (aCSF - artificial cerebrospinal fluid) into the LC of Wistar rats. Bilateral gap junction blockade in LC neurons did not affect resting ventilation; however, the increase in ventilation produced by hypercapnia (7% CO2) was reduced by ∼25% after CARB 1mM or 3mM injection (1939.7±104.8mLkg(-1)min(-1) for the aCSF group and 1468.3±122.2mLkg(-1)min(-1) for 1mM CARB, P<0.05; 1939.7±104.8mLkg(-1)min(-1) for the aCSF group and 1540.9±68.4mLkg(-1)min(-1) for the 3mM CARB group, P<0.05) due largely to a decrease in respiratory frequency. GZA injection or CARB injection outside the LC (peri-LC) had no effect on ventilation under any conditions. The results suggest that gap junctions in the LC modulate the hypercapnic ventilatory response of adult rats. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. LIMITATIONS IN THE USE OF MAGNETIC FIELDS TO EXAMINE GAP JUNCTION COMMUNICATION

    EPA Science Inventory

    OBJECTIVE: We have previously shown that gap junction communication (GJC) in mouse primary hepatocytes can be enhanced by treatment with physiological levels of melatonin, and that 45-Hz magnetic fields can eliminate this enhancement in a time-dependent manner. The objective of t...

  8. Novel Methods of Automated Quantification of Gap Junction Distribution and Interstitial Collagen Quantity from Animal and Human Atrial Tissue Sections

    PubMed Central

    Yan, Jiajie; Thomson, Justin K.; Wu, Xiaomin; Zhao, Weiwei; Pollard, Andrew E.; Ai, Xun

    2014-01-01

    Background Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies. Methods and Results Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and

  9. First-principles spin-transfer torque in CuMnAs |GaP |CuMnAs junctions

    NASA Astrophysics Data System (ADS)

    Stamenova, Maria; Mohebbi, Razie; Seyed-Yazdi, Jamileh; Rungger, Ivan; Sanvito, Stefano

    2017-02-01

    We demonstrate that an all-antiferromagnetic tunnel junction with current perpendicular to the plane geometry can be used as an efficient spintronic device with potential high-frequency operation. By using state-of-the-art density functional theory combined with quantum transport, we show that the Néel vector of the electrodes can be manipulated by spin-transfer torque. This is staggered over the two different magnetic sublattices and can generate dynamics and switching. At the same time the different magnetization states of the junction can be read by standard tunneling magnetoresistance. Calculations are performed for CuMnAs |GaP |CuMnAs junctions with different surface terminations between the antiferromagnetic CuMnAs electrodes and the insulating GaP spacer. We find that the torque remains staggered regardless of the termination, while the magnetoresistance depends on the microscopic details of the interface.

  10. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) weremore » dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.« less

  11. Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease.

    PubMed

    Burke, Shoshana; Nagajyothi, Fnu; Thi, Mia M; Hanani, Menachem; Scherer, Philipp E; Tanowitz, Herbert B; Spray, David C

    2014-11-01

    Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions. Copyright © 2014. Published by Elsevier Masson SAS.

  12. Effect of gap junctions on the firing patterns and synchrony for different external inputs in the striatal fast-spiking neuron network.

    PubMed

    Zhang, Mingming; Zhao, Zongya; He, Ping; Wang, Jue

    2014-01-01

    Gap junctions are the mechanism for striatal fast-spiking interneurons (FSIs) to interconnect with each other and play an important role in determining the physiological functioning of the FSIs. To investigate the effect of gap junctions on the firing activities and synchronization of the network for different external inputs, a simple network with least connections and a Newman-Watts small-world network were constructed. Our research shows that both properties of neural networks are related to the conductance of the gap junctions, as well as the frequency and correlation of the external inputs. The effect of gap junctions on the synchronization of network is different for inputs with different frequencies and correlations. The addition of gap junctions can promote the network synchrony in some conditions but suppress it in others, and they can inhibit the firing activities in most cases. Both the firing rate and synchronization of the network increase along with the increase of the electrical coupling strength for inputs with low frequency and high correlation. Thus, the network of coupled FSIs can act as a detector for synchronous synaptic input from cortex and thalamus.

  13. Ventilatory effects of gap junction blockade in the RTN in awake rats.

    PubMed

    Hewitt, Amy; Barrie, Rachel; Graham, Michael; Bogus, Kara; Leiter, J C; Erlichman, Joseph S

    2004-12-01

    We tested the hypothesis that carbenoxolone, a pharmacological inhibitor of gap junctions, would reduce the ventilatory response to CO(2) when focally perfused within the retrotrapezoid nucleus (RTN). We tested this hypothesis by measuring minute ventilation (V(E)), tidal volume (V(T)), and respiratory frequency (F(R)) responses to increasing concentrations of inspired CO(2) (Fi(CO(2)) = 0-8%) in rats during wakefulness. We confirmed that the RTN was chemosensitive by perfusing the RTN unilaterally with either acetazolamide (AZ; 10 microM) or hypercapnic artificial cerebrospinal fluid equilibrated with 50% CO(2) (pH approximately 6.5). Focal perfusion of AZ or hypercapnic aCSF increased V(E), V(T), and F(R) during exposure to room air. Carbenoxolone (300 microM) focally perfused into the RTN decreased V(E) and V(T) in animals <11 wk of age, but V(E) and V(T) were increased in animals >12 wk of age. Glyzyrrhizic acid, a congener of carbenoxolone, did not change V(E), V(T), or F(R) when focally perfused into the RTN. Carbenoxolone binds to the mineralocorticoid receptor, but spironolactone (10 microM) did not block the disinhibition of V(E) or V(T) in older animals when combined with carbenoxolone. Thus the RTN is a CO(2) chemosensory site in all ages tested, but the function of gap junctions in the chemosensory process varies substantially among animals of different ages: gap junctions amplify the ventilatory response to CO(2) in younger animals, but appear to inhibit the ventilatory response to CO(2) in older animals.

  14. Opposite effects of the gap junction blocker octanol on focal cerebral ischemia occluded for different durations.

    PubMed

    Ding, Wenting; Zhou, Lequan; Liu, Wei; Guan, Li; Li, Xiaoying; Liu, Haimei; Yan, Fuman; Xu, Jinwen; Zeng, Weiyong; Qiu, Min

    2014-06-01

    Protectants and executioners have been demonstrated to be used by gap junctions in focal cerebral ischemia. Certain researchers hypothesized that the opposite role of gap junctions may be associated with the injury extent, which has been demonstrated to be highly correlated with occlusion duration. In order to examine this hypothesis directly, the effects of octanol, a frequently used drug, were examined to investigate the role of gap junctions, in rats following middle cerebral artery occlusion (MCAO) for 30 min/2 h and 24 h reperfusion, respectively. Octanol significantly reduced the infarct volume following 2 h of occlusion concomitant with lower neurological deficits, whereas it enlarged the infarct volume following 30 min of occlusion. Consistently, octanol attenuated the number of transferase dUTP nick-end labeling (TUNEL) positive neurons in the hippocampal CA1 region following 2 h of occlusion, while opposite effects were observed for 30 min of occlusion. Further immunohistochemical studies demonstrated that the expression of B-cell leukemia-2 (Bcl-2, anti-apoptotic protein) was upregulated and that Bcl-2-associated X (Bax, proapoptotic protein) was downregulated following 2 h of occlusion in the octanol group compared with the ischemic group. Conversely, octanol downregulated the expression of the Bcl-2 protein concomitant with increased Bax protein following 30 min of occlusion. These results indicated that the gap junction blocker octanol can protect against ischemic injury following long-term occlusion, however, can aggravate ischemic injury following short-term occlusion.

  15. Gap Junctional Coupling in Lenses from α8 Connexin Knockout Mice

    PubMed Central

    Baldo, George J.; Gong, Xiaohua; Martinez-Wittinghan, Francisco J.; Kumar, Nalin M.; Gilula, Norton B.; Mathias, Richard T.

    2001-01-01

    Lens fiber cell gap junctions contain α3 (Cx46) and α8 (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either α3 or α8 connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from α3 connexin knockout lenses. Here, we present original data from α8 connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell–cell contact of ∼1 S/cm2, which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is ∼0.4 S/cm2, and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/−) was ∼70% of normal and 30–40% of the normal for both −/− lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of α3 +/− lenses. In contrast, the coupling of MF in the α8 +/− lenses was the same as normal. Moreover, no coupling was detected in the MF of α3 −/− lenses. Together, these results suggest that α3 connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and α3 connexin −/− lenses. However, in α8 −/− lenses, the pure α3 connexin junctions did not gate closed in the response to acidification. Since α3 connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears α8 connexin regulates the gating

  16. Gap junctional communication in the male reproductive system.

    PubMed

    Pointis, Georges; Fiorini, Céline; Defamie, Norah; Segretain, Dominique

    2005-12-20

    Male fertility is a highly controlled process that allows proliferation, meiosis and differentiation of male germ cells in the testis, final maturation in the epididymis and also requires functional male accessory glands: seminal vesicles, prostate and corpus cavernosum. In addition to classical endocrine and paracrine controls, mainly by gonadotropins LH and FSH and steroids, there is now strong evidence that all these processes are dependent upon the presence of homocellular or heterocellular junctions, including gap junctions and their specific connexins (Cxs), between the different cell types that structure the male reproductive tract. The present review is focused on the identification of Cxs, their distribution in the testis and in different structures of the male genital tract (epididymis, seminal vesicle, prostate, corpus cavernosum), their crucial role in the control of spermatogenesis and their implication in the function of the male accessory glands, including functional smooth muscle tone. Their potential dysfunctions in some testis (spermatogenic arrest, seminoma) and prostate (benign hyperplasia, adenocarcinoma) diseases and in the physiopathology of the human erectile function are also discussed.

  17. Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

    PubMed

    Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

    2014-04-01

    Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study

  18. Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

    PubMed Central

    Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.

    2015-01-01

    ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. PMID:25908859

  19. The Role of Gap Junction Communication and Oxidative Stress in the Propagation of Toxic Effects among High-Dose α-Particle-Irradiated Human Cells

    PubMed Central

    Autsavapromporn, Narongchai; de Toledo, Sonia M.; Little, John B.; Jay-Gerin, Jean-Paul; Harris, Andrew L.; Azzam, Edouard I.

    2011-01-01

    We investigated the roles of gap junction communication and oxidative stress in modulating potentially lethal damage repair in human fibroblast cultures exposed to doses of α particles or γ rays that targeted all cells in the cultures. As expected, α particles were more effective than γ rays at inducing cell killing; further, holding γ-irradiated cells in the confluent state for several hours after irradiation promoted increased survival and decreased chromosomal damage. However, maintaining α-particle-irradiated cells in the confluent state for various times prior to subculture resulted in increased rather than decreased lethality and was associated with persistent DNA damage and increased protein oxidation and lipid peroxidation. Inhibiting gap junction communication with 18-α-glycyrrhetinic acid or by knockdown of connexin43, a constitutive protein of junctional channels in these cells, protected against the toxic effects in α-particle-irradiated cell cultures during confluent holding. Upregulation of antioxidant defense by ectopic overexpression of glutathione peroxidase protected against cell killing by α particles when cells were analyzed shortly after exposure. However, it did not attenuate the decrease in survival during confluent holding. Together, these findings indicate that the damaging effect of α particles results in oxidative stress, and the toxic effects in the hours after irradiation are amplified by intercellular communication, but the communicated molecule(s) is unlikely to be a substrate of glutathione peroxidase. PMID:21388278

  20. Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes

    PubMed Central

    Connell, Jennifer Petsche; Augustini, Emily; Moise, Kenneth J; Johnson, Anthony; Jacot, Jeffrey G

    2013-01-01

    Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC. PMID:23634988

  1. Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation.

    PubMed

    Deymier, P A; Swinteck, N; Runge, K; Deymier-Black, A; Hoying, J B

    2015-01-01

    We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

  2. Effects of multipurpose solutions (MPS) for hydrogel contact lenses on gap-junctional intercellular communication (GJIC) in rabbit corneal keratocytes.

    PubMed

    Sumide, Taizo; Tsuchiya, Toshie

    2003-02-15

    To ensure the effects of multipurpose solutions (MPS) for hydrogel contact lenses on the cornea, the inhibitory activity of three types of MPS on corneal cells has been evaluated with the use of scrape loading and dye transfer assay (SLDT assay) and Western blotting on rabbit corneal keratocytes (RC4). In SLDT assay, MPS-A and poloxamine showed dose-dependent inhibitory activity, suggesting the inhibitory action of MPS-A and poloxamine to gap junctional intercellular communication (GJIC) in the tested cells. Moreover, after treatment with MPS-A, the GJIC was initially inhibited within 4 h, and thereafter gradually turned to an approximately 60% level of the initial value. When MPS-A was removed from the incubation media after exposure of the cells, the recovery of GJIC was time dependent and returned to approximately initial levels at 8 h. Complete recovery was established after approximately 24 h. These findings suggested that the inhibitory action of MPS-A on corneal keratocytes was reversible. This inhibition was accompanied by a decrease in the quantity of connexin-43, which is a major protein constituting the gap junctional channel of these cells, and its change in the phosphorylation state. Taken together, it was suggested that MPS-A interacts with connexin-43, inducing an inhibitory action on GJIC. (c) 2002 Wiley Periodicals, Inc.

  3. Gap Junctional Coupling is Essential for Epithelial Repair in the Avian Cochlea

    PubMed Central

    Nickel, Regina; Forge, Andrew

    2014-01-01

    The loss of auditory hair cells triggers repair responses within the population of nonsensory supporting cells. When hair cells are irreversibly lost from the mammalian cochlea, supporting cells expand to fill the resulting lesions in the sensory epithelium, an initial repair process that is dependent on gap junctional intercellular communication (GJIC). In the chicken cochlea (the basilar papilla or BP), dying hair cells are extruded from the epithelium and supporting cells expand to fill the lesions and then replace hair cells via mitotic and/or conversion mechanisms. Here, we investigated the involvement of GJIC in the initial epithelial repair process in the aminoglycoside-damaged BP. Gentamicin-induced hair cell loss was associated with a decrease of chicken connexin43 (cCx43) immunofluorescence, yet cCx30-labeled gap junction plaques remained. Fluorescence recovery after photobleaching experiments confirmed that the GJIC remained robust in gentamicin-damaged explants, but regionally asymmetric coupling was no longer evident. Dye injections in slice preparations from undamaged BP explants identified cell types with characteristic morphologies along the neural-abneural axis, but these were electrophysiologically indistinct. In gentamicin-damaged BP, supporting cells expanded to fill space formerly occupied by hair cells and displayed more variable electrophysiological phenotypes. When GJIC was inhibited during the aminoglycoside damage paradigm, the epithelial repair response halted. Dying hair cells were retained within the sensory epithelium and supporting cells remained unexpanded. These observations suggest that repair of the auditory epithelium shares common mechanisms across vertebrate species and emphasize the importance of functional gap junctions in maintaining a homeostatic environment permissive for subsequent hair cell regeneration. PMID:25429127

  4. INFLUENCE OF SODIUM ARSENITE ON GAP JUNCTION COMMUNICATION IN RAT LIVER EPITHELIAL CELLS

    EPA Science Inventory

    Influence of sodium arsenite on gap junction communication in rat-Iiver epitheiial cells.

    Arsenic is known to cause certain types of cancers, hepatitis, cirrhosis and neurological disorders as well as cardiovascular and reproductive effects and skin lesions. The mechanism...

  5. In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

    PubMed

    Nayak, Bibhukalyan Prasad; Goh, James Cho Hong; Toh, Siew Lok; Satpathy, Gyan Ranjan

    2010-03-01

    Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. To replace a severely damaged ligament, a tissue-engineered graft preinstalled with interfacial fibrocartilage, which is being regenerated from stem cells, appears to be more promising than ligament-alone graft. Such a concept can be realized by a biomimetic approach of establishing a dynamic communication of stem cells with bone cells and/or ligament fibroblasts in vitro. The current study has two objectives. The first objective is to demonstrate functional coculture of bone marrow-derived stem cells (BMSCs) with mature bone cells/ligament fibroblasts as evidenced by gap-junctional communication in vitro. The second objective is to investigate the role of BMSCs in the regeneration of fibrocartilage within the coculture. Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control). Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control. The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration. The results open up a path to envisage a composite

  6. Fabricating Atom-Sized Gaps by Field-Aided Atom Migration in Nanoscale Junctions

    NASA Astrophysics Data System (ADS)

    Liu, Ran; Bi, Jun-Jie; Xie, Zhen; Yin, Kaikai; Wang, Dunyou; Zhang, Guang-Ping; Xiang, Dong; Wang, Chuan-Kui; Li, Zong-Liang

    2018-05-01

    The gap sizes between electrodes generated by typical methods are generally much larger than the dimension of a common molecule when fabricating a single-molecule junction, which dramatically suppresses the yield of single-molecule junctions. Based on the ab initio calculations, we develop a strategy named the field-aided method to accurately fabricate an atomic-sized gap between gold nanoelectrodes. To understand the mechanism of this strategy, configuration evolutions of gold nanojunction in stretching and compressing processes are calculated. The numerical results show that, in the stretching process, the gold atoms bridged between two electrodes are likely to form atomic chains. More significantly, lattice vacant positions can be easily generated in stretching and compressing processes, which make field-aided gap generation possible. In field-aided atom migration (FAAM), the external field can exert driving force, enhance the initial energy of the system, and decrease the barrier in the migration path, which makes the atom migration feasible. Conductance and stretching and compressing forces, as measurable variables in stretching and compressing processes, present very useful signals for determining the time to perform FAAM. Following this desirable strategy, we successfully fabricate gold nanogaps with a dimension of 0.38 ±0.05 nm in the experiment, as our calculation simulates.

  7. Blockade of brain stem gap junctions increases phrenic burst frequency and reduces phrenic burst synchronization in adult rat.

    PubMed

    Solomon, Irene C; Chon, Ki H; Rodriguez, Melissa N

    2003-01-01

    Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1-5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 microM), 18alpha-glycyrrhetinic acid (25-100 microM), 18beta-glycyrrhetinic acid (25-100 microM), octanol (200-300 microM), or heptanol (200 microM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (~95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge (P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T(I) (P < 0.01) and T(E) (P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a "bell-shaped" or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (> or =130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to

  8. Homotypic gap junctional communication associated with metastasis increases suppression increases with PKA kinase activity and is unaffected by P13K inhibition

    USDA-ARS?s Scientific Manuscript database

    Loss of gap junctional intercellular communication (GJIC) between cancer cells is a common characteristic of malignant transformation. This communication is mediated by connexin proteins that make up the functional units of gap junctions. Connexins are highly regulated at the protein level and phosp...

  9. Cellular Interaction of Integrin α3β1 with Laminin 5 Promotes Gap Junctional Communication

    PubMed Central

    Lampe, Paul D.; Nguyen, Beth P.; Gil, Susana; Usui, Marcia; Olerud, John; Takada, Yoshikazu; Carter, William G.

    1998-01-01

    Wounding of skin activates epidermal cell migration over exposed dermal collagen and fibronectin and over laminin 5 secreted into the provisional basement membrane. Gap junctional intercellular communication (GJIC) has been proposed to integrate the individual motile cells into a synchronized colony. We found that outgrowths of human keratinocytes in wounds or epibole cultures display parallel changes in the expression of laminin 5, integrin α3β1, E-cadherin, and the gap junctional protein connexin 43. Adhesion of keratinocytes on laminin 5, collagen, and fibronectin was found to differentially regulate GJIC. When keratinocytes were adhered on laminin 5, both structural (assembly of connexin 43 in gap junctions) and functional (dye transfer) assays showed a two- to threefold increase compared with collagen and five- to eightfold over fibronectin. Based on studies with immobilized integrin antibody and integrin-transfected Chinese hamster ovary cells, the interaction of integrin α3β1 with laminin 5 was sufficient to promote GJIC. Mapping of intermediate steps in the pathway linking α3β1–laminin 5 interactions to GJIC indicated that protein trafficking and Rho signaling were both required. We suggest that adhesion of epithelial cells to laminin 5 in the basement membrane via α3β1 promotes GJIC that integrates individual cells into synchronized epiboles. PMID:9852164

  10. TWO INNEXINS OF Spodoptera litura INFLUENCES HEMICHANNEL AND GAP JUNCTION FUNCTIONS IN CELLULAR IMMUNE RESPONSES.

    PubMed

    Pang, Zunyu; Li, Ming; Yu, Dongshuai; Yan, Zhang; Liu, Xinyi; Ji, Xinglai; Yang, Yang; Hu, Jiansheng; Luo, Kaijun

    2015-09-01

    Insect cellular immune responses include encapsulation, nodule formation, and phagocytosis. Hemichannels and gap junctions are involved in these cellular actions. Innexins (Inxs: analogous to the vertebrate connexins) form hemichannels and gap junctions, but the molecular mechanisms underlying their biology is still unclear. In this article, we reported a steady-state level of Inxs (SpliInxs) in hemocytes of Spodoptera litura, which formed nonfunctional hemichannels on the cell surface to maintain normal metabolism. We also reported that two innnexins (SpliInx2 and SpliInx3) were expressed significantly higher in hemocytes compared to other tissues, suggesting that they play important roles in hemocytes. Amino acid analysis found that two cysteine residues in two extracellular loops provided the capability for SpliInx2 and SpliInx3 hemichannels to dock into gap junctions. Western blotting demonstrated that both extracellular and intracellular loops of SpliInx3 and the extracellular loops of SpliInx2 might undergo posttranslational modification during the formation of a steady-state hemichannel. During hemichannel formation, SpliInx2 presented as one isoform, while SpliInx3 presented as three isoforms. These results provide fundamental knowledge for further study of how steady-state levels of SpliInxs are dynamically adjusted to perform cellular immune responses under immune challenge. © 2015 Wiley Periodicals, Inc.

  11. 2',5'-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation.

    PubMed

    Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho

    2002-09-30

    We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

  12. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Du, Kuo; Williams, C. David; McGill, Mitchell R.

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented whenmore » animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2

  13. Pregnancy-induced remodelling and enhanced endothelium-derived hyperpolarization-type vasodilator activity in rat uterine radial artery: transient receptor potential vanilloid type 4 channels, caveolae and myoendothelial gap junctions

    PubMed Central

    Senadheera, Sevvandi; Bertrand, Paul P; Grayson, T Hilton; Leader, Leo; Murphy, Timothy V; Sandow, Shaun L

    2013-01-01

    In pregnancy, the vasculature of the uterus undergoes rapid remodelling to increase blood flow and maintain perfusion to the fetus. The present study determines the distribution and density of caveolae, transient receptor potential vanilloid type 4 channels (TRPV4) and myoendothelial gap junctions, and the relative contribution of related endothelium-dependent vasodilator components in uterine radial arteries of control virgin non-pregnant and 20-day late-pregnant rats. The hypothesis examined is that specific components of endothelium-dependent vasodilator mechanisms are altered in pregnancy-related uterine radial artery remodelling. Conventional and serial section electron microscopy were used to determine the morphological characteristics of uterine radial arteries from control and pregnant rats. TRPV4 distribution and expression was examined using conventional confocal immunohistochemistry, and the contribution of endothelial TRPV4, nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-type activity determined using pressure myography with pharmacological intervention. Data show outward hypertrophic remodelling occurs in uterine radial arteries in pregnancy. Further, caveolae density in radial artery endothelium and smooth muscle from pregnant rats was significantly increased by ∼94% and ∼31%, respectively, compared with control, whereas caveolae density did not differ in endothelium compared with smooth muscle from control. Caveolae density was significantly higher by ∼59% on the abluminal compared with the luminal surface of the endothelium in uterine radial artery of pregnant rats but did not differ at those surfaces in control. TRPV4 was present in endothelium and smooth muscle, but not associated with internal elastic lamina hole sites in radial arteries. TRPV4 fluorescence intensity was significantly increased in the endothelium and smooth muscle of radial artery of pregnant compared with control rats by ∼2.6- and 5.5-fold

  14. Integrating evolutionary game theory into an agent-based model of ductal carcinoma in situ: Role of gap junctions in cancer progression.

    PubMed

    Malekian, Negin; Habibi, Jafar; Zangooei, Mohammad Hossein; Aghakhani, Hojjat

    2016-11-01

    There are many cells with various phenotypic behaviors in cancer interacting with each other. For example, an apoptotic cell may induce apoptosis in adjacent cells. A living cell can also protect cells from undergoing apoptosis and necrosis. These survival and death signals are propagated through interaction pathways between adjacent cells called gap junctions. The function of these signals depends on the cellular context of the cell receiving them. For instance, a receiver cell experiencing a low level of oxygen may interpret a received survival signal as an apoptosis signal. In this study, we examine the effect of these signals on tumor growth. We make an evolutionary game theory component in order to model the signal propagation through gap junctions. The game payoffs are defined as a function of cellular context. Then, the game theory component is integrated into an agent-based model of tumor growth. After that, the integrated model is applied to ductal carcinoma in situ, a type of early stage breast cancer. Different scenarios are explored to observe the impact of the gap junction communication and parameters of the game theory component on cancer progression. We compare these scenarios by using the Wilcoxon signed-rank test. The Wilcoxon signed-rank test succeeds in proving a significant difference between the tumor growth of the model before and after considering the gap junction communication. The Wilcoxon signed-rank test also proves that the tumor growth significantly depends on the oxygen threshold of turning survival signals into apoptosis. In this study, the gap junction communication is modeled by using evolutionary game theory to illustrate its role at early stage cancers such as ductal carcinoma in situ. This work indicates that the gap junction communication and the oxygen threshold of turning survival signals into apoptosis can notably affect cancer progression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Simvastatin-induced up-regulation of gap junctions composed of connexin 43 sensitize Leydig tumor cells to etoposide: an involvement of PKC pathway.

    PubMed

    Wang, Lingzhi; Fu, Yanni; Peng, Jianxin; Wu, Dengpan; Yu, Meiling; Xu, Chengfang; Wang, Qin; Tao, Liang

    2013-10-04

    Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    PubMed Central

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

  17. Localization of connexin 43 gap junctions and hemichannels in tanycytes of adult mice.

    PubMed

    Szilvásy-Szabó, Anett; Varga, Edina; Beliczai, Zsuzsa; Lechan, Ronald M; Fekete, Csaba

    2017-10-15

    Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of β-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Sputtered Metal Oxide Broken Gap Junctions for Tandem Solar Cells

    NASA Astrophysics Data System (ADS)

    Johnson, Forrest

    Broken gap metal oxide junctions have been created for the first time by sputtering using ZnSnO3 for the n-type material and Cu 2O or CuAlO2 for the p-type material. Films were sputtered from either ceramic or metallic targets at room temperature from 10nm to 220nm thick. The band structure of the respective materials have theoretical work functions which line up with the band structure for tandem CIAGS/CIGS solar cell applications. Multiple characterization methods demonstrated consistent ohmic I-V profiles for devices on rough surfaces such as ITO/glass and a CIAGS cell. Devices with total junction specific contact resistance of under 0.001 Ohm-cm2 have been achieved with optical transmission close to 100% using 10nm films. Devices showed excellent stability up to 600°C anneals over 1hr using ZnSnO3 and CuAlO2. These films were also amorphous -a great diffusion barrier during top cell growth at high temperatures. Rapid Thermal Anneal (RTA) demonstrated the ability to shift the band structure of the whole device, allowing for tuning it to align with adjacent solar layers. These results remove a key barrier for mass production of multi-junction thin film solar cells.

  19. Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

    PubMed Central

    Kuras, Paulina; Lydka-Zarzycka, Marta; Bilinska, Barbara

    2013-01-01

    In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP) on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43), in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w.) resulted in the reduction of junction proteins expressions (P < 0.05, P < 0.01) and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P < 0.05), N-cadherin, and β-catenin (statistically nonsignificant) levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway. PMID:23737770

  20. BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells.

    PubMed

    Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R; Trimmer, James S; Rasband, Matthew N; Misonou, Hiroaki

    2015-05-06

    In myelinated axons, K(+) channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na(+) channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K(+) channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K(+) channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni(2+) elicited a similar effect on APs, indicating the involvement of Ni(2+)-sensitive Ca(2+) channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. Copyright © 2015 the authors 0270-6474/15/357082-13$15.00/0.

  1. Alterations in gap junction connexin43/connexin45 ratio mediate a transition from quiescence to excitation in a mathematical model of the myometrium

    PubMed Central

    Sheldon, Rachel E.; Mashayamombe, Chipo; Shi, Shao-Qing; Garfield, Robert E.; Shmygol, Anatoly; Blanks, Andrew M.; van den Berg, Hugo A.

    2014-01-01

    The smooth muscle cells of the uterus contract in unison during delivery. These cells achieve coordinated activity via electrical connections called gap junctions which consist of aggregated connexin proteins such as connexin43 and connexin45. The density of gap junctions governs the excitability of the myometrium (among other factors). An increase in gap junction density occurs immediately prior to parturition. We extend a mathematical model of the myometrium by incorporating the voltage-dependence of gap junctions that has been demonstrated in the experimental literature. Two functional subtypes exist, corresponding to systems with predominantly connexin43 and predominantly connexin45, respectively. Our simulation results indicate that the gap junction protein connexin45 acts as a negative modulator of uterine excitability, and hence, activity. A network with a higher proportion of connexin45 relative to connexin43 is unable to excite every cell. Connexin45 has much more rapid gating kinetics than connexin43 which we show limits the maximum duration of a local burst of activity. We propose that this effect regulates the degree of synchronous excitation attained during a contraction. Our results support the hypothesis that as labour approaches, connexin45 is downregulated to allow action potentials to spread more readily through the myometrium. PMID:25401181

  2. CRTC2 and Nedd4 ligase involvement in FSH and TGFβ1 upregulation of connexin43 gap junction.

    PubMed

    Fang, Wei-Ling; Lai, Si-Yi; Lai, Wei-An; Lee, Ming-Ting; Liao, Ching-Fong; Ke, Ferng-Chun; Hwang, Jiuan-Jiuan

    2015-12-01

    The major mission of the ovarian follicle is the timely production of the mature fertilizable oocyte, and this is achieved by gonadotropin-regulated, gap junction-mediated cell-cell communication between the oocyte and surrounding nurturing granulosa cells. We have demonstrated that FSH and transforming growth factor beta 1 (TGFβ1) stimulate Gja1 gene-encoded connexin43 (Cx43) gap junction formation/function in rat ovarian granulosa cells is important for their induction of steroidogenesis; additionally, cAMP-protein kinase A (PKA)- and calcium-calcineurin-sensitive cAMP response element-binding (CREB) coactivator CRTC2 plays a crucial role during steroidogenesis. This study was to explore the potential molecular mechanism whereby FSH and TGFβ1 regulate Cx43 synthesis and degradation, particularly the involvement of CRTC2 and ubiquitin ligase Nedd4. Primary culture of granulosa cells from ovarian antral follicles of gonadotropin-primed immature rats was used. At 48 h post-treatment, FSH plus TGFβ1 increased Cx43 level and gap junction function in a PKA- and calcineurin-dependent manner, and TGFβ1 acting through its type I receptor modulated FSH action. Chromatin-immunoprecipitation analysis reveals FSH induced an early-phase (45 min) and FSH+TGFβ1 further elicited a late-phase (24 h) increase in CRTC2, CREB and CBP binding to the Gja1 promoter. Additionally, FSH+TGFβ1 increased the half-life of hyper-phosphorylated Cx43 (Cx43-P2). Also, the proteasome inhibitor MG132 prevented the brefeldin A (blocker of protein transport through Golgi)-reduced Cx43-P2 level and membrane Cx43 gap junction plaque. This is associated with FSH+TGFβ1-attenuated Cx43 interaction with Nedd4 and Cx43 ubiquitination. In all, this study uncovers that FSH and TGFβ1 upregulation of Cx43 gap junctions in ovarian granulosa cells critically involves enhancing CRTC2/CREB/CBP-mediated Cx43 expression and attenuating ubiquitin ligase Nedd4-mediated proteosomal degradation of Cx43 protein

  3. Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

    PubMed

    Kim, D Y; Kam, Y; Koo, S K; Joe, C O

    1999-02-26

    The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

  4. RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer.

    PubMed

    Zihni, Ceniz; Terry, Stephen James

    2015-07-01

    The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.

    PubMed

    Oh, Hyun-Woo; Campusano, Jorge M; Hilgenberg, Lutz G W; Sun, Xicui; Smith, Martin A; O'Dowd, Diane K

    2008-02-15

    Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.

  6. Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish

    PubMed Central

    Irion, Uwe; Frohnhöfer, Hans Georg; Krauss, Jana; Çolak Champollion, Tuǧba; Maischein, Hans-Martin; Geiger-Rudolph, Silke; Weiler, Christian; Nüsslein-Volhard, Christiane

    2014-01-01

    Interactions between all three pigment cell types are required to form the stripe pattern of adult zebrafish (Danio rerio), but their molecular nature is poorly understood. Mutations in leopard (leo), encoding Connexin 41.8 (Cx41.8), a gap junction subunit, cause a phenotypic series of spotted patterns. A new dominant allele, leotK3, leads to a complete loss of the pattern, suggesting a dominant negative impact on another component of gap junctions. In a genetic screen, we identified this component as Cx39.4 (luchs). Loss-of-function alleles demonstrate that luchs is required for stripe formation in zebrafish; however, the fins are almost not affected. Double mutants and chimeras, which show that leo and luchs are only required in xanthophores and melanophores, but not in iridophores, suggest that both connexins form heteromeric gap junctions. The phenotypes indicate that these promote homotypic interactions between melanophores and xanthophores, respectively, and those cells instruct the patterning of the iridophores. DOI: http://dx.doi.org/10.7554/eLife.05125.001 PMID:25535837

  7. Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43.

    PubMed

    Heyman, Nathanael S; Kurjiaka, David T; Ek Vitorin, Jose F; Burt, Janis M

    2009-07-01

    Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.

  8. Progressive age-dependence and frequency difference in the effect of gap junctions on active cochlear amplification and hearing.

    PubMed

    Zong, Liang; Chen, Jin; Zhu, Yan; Zhao, Hong-Bo

    2017-07-22

    Mutations of Connexin 26 (Cx26, GJB2), which is a predominant gap junction isoform in the cochlea, can induce high incidence of nonsyndromic hearing loss. We previously found that targeted-deletion of Cx26 in supporting Deiters cells and outer pillar cells in the cochlea can influence outer hair cell (OHC) electromotility and reduce active cochlear amplification leading to hearing loss, even though there are no gap junction connexin expressions in the auditory sensory hair cells. Here, we further report that hearing loss and the reduction of active amplification in the Cx26 targeted-deletion mice are progressive and different at high and low frequency regions, first occurring in the high frequency region and then progressively extending to the middle and low frequency regions with mouse age increased. The speed of hearing loss extending was fast in the basal high frequency region and slow in the apical low frequency region, showing a logarithmic function with mouse age. Before postnatal day 25, there were no significant hearing loss and the reduction of active cochlear amplification in the low frequency region. Hearing loss and the reduction of active cochlear amplification also had frequency difference, severe and large in the high frequency regions. These new data indicate that the effect of gap junction on active cochlear amplification is progressive, but, consistent with our previous report, exists in both high and low frequency regions in adulthood. These new data also suggest that cochlear gap junctions may have an important role in age-related hearing loss. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Multi-junction, monolithic solar cell using low-band-gap materials lattice matched to GaAs or Ge

    DOEpatents

    Olson, Jerry M.; Kurtz, Sarah R.; Friedman, Daniel J.

    2001-01-01

    A multi-junction, monolithic, photovoltaic solar cell device is provided for converting solar radiation to photocurrent and photovoltage with improved efficiency. The solar cell device comprises a plurality of semiconductor cells, i.e., active p/n junctions, connected in tandem and deposited on a substrate fabricated from GaAs or Ge. To increase efficiency, each semiconductor cell is fabricated from a crystalline material with a lattice constant substantially equivalent to the lattice constant of the substrate material. Additionally, the semiconductor cells are selected with appropriate band gaps to efficiently create photovoltage from a larger portion of the solar spectrum. In this regard, one semiconductor cell in each embodiment of the solar cell device has a band gap between that of Ge and GaAs. To achieve desired band gaps and lattice constants, the semiconductor cells may be fabricated from a number of materials including Ge, GaInP, GaAs, GaInAsP, GaInAsN, GaAsGe, BGaInAs, (GaAs)Ge, CuInSSe, CuAsSSe, and GaInAsNP. To further increase efficiency, the thickness of each semiconductor cell is controlled to match the photocurrent generated in each cell. To facilitate photocurrent flow, a plurality of tunnel junctions of low-resistivity material are included between each adjacent semiconductor cell. The conductivity or direction of photocurrent in the solar cell device may be selected by controlling the specific p-type or n-type characteristics for each active junction.

  10. Distinct molecular targets including SLO-1 and gap junctions are engaged across a continuum of ethanol concentrations in Caenorhabditis elegans

    PubMed Central

    Dillon, James; Andrianakis, Ioannis; Mould, Richard; Ient, Ben; Liu, Wei; James, Christopher; O'Connor, Vincent; Holden-Dye, Lindy

    2013-01-01

    Ethanol (alcohol) interacts with diverse molecular effectors across a range of concentrations in the brain, eliciting intoxication through to sedation. Invertebrate models including the nematode worm Caenorhabditis elegans have been deployed for molecular genetic studies to inform on key components of these alcohol signaling pathways. C. elegans studies have typically employed external dosing with high (>250 mM) ethanol concentrations: A careful analysis of responses to low concentrations is lacking. Using the C. elegans pharyngeal system as a paradigm, we report a previously uncharacterized continuum of cellular and behavioral responses to ethanol from low (10 mM) to high (300 mM) concentrations. The complexity of these responses indicates that the pleiotropic action of ethanol observed in mammalian brain is conserved in this invertebrate model. We investigated two candidate ethanol effectors, the calcium-activated K+ channel SLO-1 and gap junctions, and show that they contribute to, but are not sole determinants of, the low- and high-concentration effects, respectively. Notably, this study shows cellular and whole organismal behavioral responses to ethanol in C. elegans that directly equate to intoxicating through to supralethal blood alcohol concentrations in humans and provides an important benchmark for interpretation of paradigms that seek to inform on human alcohol use disorders.—Dillon, J., Andrianakis, I., Mould, R., Ient, B., Liu, W., James, C., O'Connor, V., Holden-Dye, L. Distinct molecular targets including SLO-1 and gap junctions are engaged across a continuum of ethanol concentrations in Caenorhabditis elegans. PMID:23882127

  11. Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

    PubMed

    Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

    2012-03-09

    In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. High performance as-grown and annealed high band gap tunnel junctions: Te behavior at the interface

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bedair, S. M., E-mail: bedair@ncsu.edu; Harmon, Jeffrey L.; Carlin, C. Zachary

    2016-05-16

    The performance of n{sup +}-InGaP(Te)/p{sup +}-AlGaAs(C) high band gap tunnel junctions (TJ) is critical for achieving high efficiency in multijunction photovoltaics. Several limitations for as grown and annealed TJ can be attributed to the Te doping of InGaP and its behavior at the junction interface. Te atoms in InGaP tend to get attached at step edges, resulting in a Te memory effect. In this work, we use the peak tunneling current (J{sub pk}) in this TJ as a diagnostic tool to study the behavior of the Te dopant at the TJ interface. Additionally, we used our understanding of Te behaviormore » at the interface, guided by device modeling, to modify the Te source shut-off procedure and the growth rate. These modifications lead to a record performance for both the as-grown (2000 A/cm{sup 2}) and annealed (1000 A/cm{sup 2}) high band gap tunnel junction.« less

  13. The Role of Gap Junction Channels During Physiologic and Pathologic Conditions of the Human Central Nervous System

    PubMed Central

    Basilio, Daniel; Sáez, Juan C.; Orellana, Juan A.; Raine, Cedric S.; Bukauskas, Feliksas; Bennett, Michael V. L.; Berman, Joan W.

    2013-01-01

    Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP3, and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system. PMID:22438035

  14. Gating of Connexin Channels by transjunctional-voltage: Conformations and models of open and closed states.

    PubMed

    Bargiello, Thaddeus A; Oh, Seunghoon; Tang, Qingxiu; Bargiello, Nicholas K; Dowd, Terry L; Kwon, Taekyung

    2018-01-01

    Voltage is an important physiologic regulator of channels formed by the connexin gene family. Connexins are unique among ion channels in that both plasma membrane inserted hemichannels (undocked hemichannels) and intercellular channels (aggregates of which form gap junctions) have important physiological roles. The hemichannel is the fundamental unit of gap junction voltage-gating. Each hemichannel displays two distinct voltage-gating mechanisms that are primarily sensitive to a voltage gradient formed along the length of the channel pore (the transjunctional voltage) rather than sensitivity to the absolute membrane potential (V m or V i-o ). These transjunctional voltage dependent processes have been termed V j - or fast-gating and loop- or slow-gating. Understanding the mechanism of voltage-gating, defined as the sequence of voltage-driven transitions that connect open and closed states, first and foremost requires atomic resolution models of the end states. Although ion channels formed by connexins were among the first to be characterized structurally by electron microscopy and x-ray diffraction in the early 1980's, subsequent progress has been slow. Much of the current understanding of the structure-function relations of connexin channels is based on two crystal structures of Cx26 gap junction channels. Refinement of crystal structure by all-atom molecular dynamics and incorporation of charge changing protein modifications has resulted in an atomic model of the open state that arguably corresponds to the physiologic open state. Obtaining validated atomic models of voltage-dependent closed states is more challenging, as there are currently no methods to solve protein structure while a stable voltage gradient is applied across the length of an oriented channel. It is widely believed that the best approach to solve the atomic structure of a voltage-gated closed ion channel is to apply different but complementary experimental and computational methods and to use

  15. Gap junctions mediate large-scale Turing structures in a mean-field cortex driven by subcortical noise

    NASA Astrophysics Data System (ADS)

    Steyn-Ross, Moira L.; Steyn-Ross, D. A.; Wilson, M. T.; Sleigh, J. W.

    2007-07-01

    One of the grand puzzles in neuroscience is establishing the link between cognition and the disparate patterns of spontaneous and task-induced brain activity that can be measured clinically using a wide range of detection modalities such as scalp electrodes and imaging tomography. High-level brain function is not a single-neuron property, yet emerges as a cooperative phenomenon of multiply-interacting populations of neurons. Therefore a fruitful modeling approach is to picture the cerebral cortex as a continuum characterized by parameters that have been averaged over a small volume of cortical tissue. Such mean-field cortical models have been used to investigate gross patterns of brain behavior such as anesthesia, the cycles of natural sleep, memory and erasure in slow-wave sleep, and epilepsy. There is persuasive and accumulating evidence that direct gap-junction connections between inhibitory neurons promote synchronous oscillatory behavior both locally and across distances of some centimeters, but, to date, continuum models have ignored gap-junction connectivity. In this paper we employ simple mean-field arguments to derive an expression for D2 , the diffusive coupling strength arising from gap-junction connections between inhibitory neurons. Using recent neurophysiological measurements reported by Fukuda [J. Neurosci. 26, 3434 (2006)], we estimate an upper limit of D2≈0.6cm2 . We apply a linear stability analysis to a standard mean-field cortical model, augmented with gap-junction diffusion, and find this value for the diffusive coupling strength to be close to the critical value required to destabilize the homogeneous steady state. Computer simulations demonstrate that larger values of D2 cause the noise-driven model cortex to spontaneously crystalize into random mazelike Turing structures: centimeter-scale spatial patterns in which regions of high-firing activity are intermixed with regions of low-firing activity. These structures are consistent with the

  16. Gap junctions between accessory medulla neurons appear to synchronize circadian clock cells of the cockroach Leucophaea maderae.

    PubMed

    Schneider, Nils-Lasse; Stengl, Monika

    2006-03-01

    The temporal organization of physiological and behavioral states is controlled by circadian clocks in apparently all eukaryotic organisms. In the cockroach Leucophaea maderae lesion and transplantation studies located the circadian pacemaker in the accessory medulla (AMe). The AMe is densely innervated by gamma-aminobutyric acid (GABA)-immunoreactive and peptidergic neurons, among them the pigment-dispersing factor immunoreactive circadian pacemaker candidates. The large majority of cells of the cockroach AMe spike regularly and synchronously in the gamma frequency range of 25-70 Hz as a result of synaptic and nonsynaptic coupling. Although GABAergic coupling forms assemblies of phase-locked cells, in the absence of synaptic release the cells remain synchronized but fire now at a stable phase difference. To determine whether these coupling mechanisms of AMe neurons, which are independent of synaptic release, are based on electrical synapses between the circadian pacemaker cells the gap-junction blockers halothane, octanol, and carbenoxolone were used in the presence and absence of synaptic transmission. Here, we show that different populations of AMe neurons appear to be coupled by gap junctions to maintain synchrony at a stable phase difference. This synchronization by gap junctions is a prerequisite to phase-locked assembly formation by synaptic interactions and to synchronous gamma-type action potential oscillations within the circadian clock.

  17. Dual-channel current valve in a three terminal zigzag graphene nanoribbon junction

    NASA Astrophysics Data System (ADS)

    Zhang, L.

    2017-02-01

    We theoretically propose a dual-channel current valve based on a three terminal zigzag graphene nanoribbon (ZGNR) junction driven by three asymmetric time-dependent pumping potentials. By means of the Keldysh Green’s function method, we show that two asymmetric charge currents can be pumped in the different left-right terminals of the device at a zero bias, which mainly stems from the single photon-assisted pumping approximation and the valley valve effect in a ZGNR p-n junction. The ON and OFF states of pumped charge currents are crucially dependent on the even-odd chain widths of the three electrodes, the pumping frequency, the lattice potential and the Fermi level. Two-tunneling spin valves are also considered to spatially separate and detect 100% polarized spin currents owing to the combined spin pump effect and the valley selective transport in a three terminal ZGNR ferromagnetic junction. Our investigations might be helpful to control the spatial and spin degrees of freedom of electrons in graphene pumping devices.

  18. Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

    PubMed

    Beyer, Eric C; Lipkind, Gregory M; Kyle, John W; Berthoud, Viviana M

    2012-08-01

    The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011. Published by Elsevier B.V.

  19. Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.

    PubMed

    Han, Jin; Kim, Bokyoung; Shin, Jung-Youn; Ryu, Seungmi; Noh, Myungkyung; Woo, Jongsu; Park, Jin-Sil; Lee, Youjin; Lee, Nohyun; Hyeon, Taeghwan; Choi, Donghoon; Kim, Byung-Soo

    2015-03-24

    Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies.

  20. Control of neuronal morphology and connectivity: emerging developmental roles for gap junctional proteins.

    PubMed

    Baker, Michael W; Macagno, Eduardo R

    2014-04-17

    Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Fractional Josephson vortices in two-gap superconductor long Josephson junctions

    NASA Astrophysics Data System (ADS)

    Kim, Ju

    2014-03-01

    We investigated the phase dynamics of long Josephson junctions (LJJ) with two-gap superconductors in the broken time reversal symmetry state. In this LJJ, spatial phase textures (i-solitons) can be excited due to the presence of two condensates and the interband Joesphson effect between them. The presence of a spatial phase texture in each superconductor layer leads to a spatial variation of the critical current density between the superconductor layers. We find that this spatial dependence of the crtitical current density can self-generate magnetic flux in the insulator layer, resulting in Josephson vortices with fractional flux quanta. Similar to the situation in a YBa2 Cu3O7 - x superconductor film grain boundary, the fractionalization of a Josephson vortex arises as a response to either periodic or random excitation of i-solitions. This suggests that magnetic flux measurements may be used to probe i-soliton excitations in multi-gap superconductor LJJs.

  2. Connexin-36 gap junctions regulate in vivo first- and second-phase insulin secretion dynamics and glucose tolerance in the conscious mouse.

    PubMed

    Head, W Steven; Orseth, Meredith L; Nunemaker, Craig S; Satin, Leslie S; Piston, David W; Benninger, Richard K P

    2012-07-01

    Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36(-/-)) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36(-/-) and control mice. Our results show that Cx36(-/-) mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36(-/-) mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36(-/-) islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.

  3. [Effects of gap junction blocking on the oxygen partial pressure in acupoints of the bladder meridian].

    PubMed

    Wang, Qi; Yu, Wei-Chang; Jiang, Hong-Zhi; Chen, Sheng-Li; Zhang, Ming-Min; Kong, E-Sheng; Huang, Guang-Ying

    2010-12-01

    To explore the relation between gap junction and meridian phenomenon. The oxygen partial pressure in acupoints [see text for formula] and in their corresponding non-acupoints of the Bladder Meridian was observed with the needle-type tissue oxygen tension sensor in the gap junction blocking goats by 1-Heptanol injection and the Connexin 43 (Cx43) gene knockout mice. (1) The oxygen partial pressure in acupoints of Bladder Meridian on goats was higher than that in non-acupoints after 1-Heptanol injection with significant differences between them (both P < 0.01). (2) The oxygen partial pressure in acupoints of Bladder Meridian on goats increased significantly after injecting 1-Heptanol as compare with that either injecting normal saline or injecting nothing with significant differences between them (all P < 0.01). (3) The oxygen partial pressure in acupoints of the Bladder Meridian was significantly higher than that in the non-acupoint controls in Cx43 wild type (WT) mice (all P < 0.01). In Cx43 heterozygote (HT) mice, the oxygen partial pressure between acupoints and non-acupoint controls showed no significant differences (all P > 0.05). (4) In acupoints, the oxygen partial pressure in Cx43 WT mice was significantly higher than that in Cx43 HT mice (all P < 0.05), while in the corresponding non-acupoints, this difference had no statistically significant (all P > 0.05). Gap junction maybe the essential factor in signal transduction of acupuncture.

  4. Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

    PubMed Central

    Stanley, Rachael L; Fleck, Roland A; Becker, David L; Goodship, Allen E; Ralphs, Jim R; Patterson-Kane, Janet C

    2007-01-01

    Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1–6 months), 5 young adults (2–7 years) and 5 old horses (18–33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury. PMID:17848160

  5. Transport properties and device-design of Z-shaped MoS2 nanoribbon planar junctions

    NASA Astrophysics Data System (ADS)

    Zhang, Hua; Zhou, Wenzhe; Liu, Qi; Yang, Zhixiong; Pan, Jiangling; Ouyang, Fangping; Xu, Hui

    2017-09-01

    Based on MoS2 nanoribbons, metal-semiconductor-metal planar junction devices were constructed. The electronic and transport properties of the devices were studied by using density function theory (DFT) and nonequilibrium Green's functions (NEGF). It is found that a band gap about 0.4 eV occurs in the planar junction. The electron and hole transmissions of the devices are mainly contributed by the Mo atomic orbitals. The electron transport channel is located at the edge of armchair MoS2 nanoribbon, while the hole transport channel is delocalized in the channel region. The I-V curve of the two-probe device shows typical transport behavior of Schottky barrier, and the threshold voltage is of about 0.2 V. The field effect transistors (FET) based on the planar junction turn out to be good bipolar transistors, the maximum current on/off ratio can reach up to 1 × 104, and the subthreshold swing is 243 mV/dec. It is found that the off-state current is dependent on the length and width of the channel, while the on-state current is almost unaffected. The switching performance of the FET is improved with increasing the length of the channel, and shows oscillation behavior with the change of the channel width.

  6. Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture.

    PubMed

    Zündorf, Gregor; Kahlert, Stefan; Reiser, Georg

    2007-07-01

    The beneficial or detrimental role of gap junction communication in the pathophysiology of brain injury is still controversial. We used co-cultures of hippocampal astrocytes and neurons, where we identified homocellular astrocyte-astrocyte and heterocellular astrocyte-neuron coupling by fluorescence recovery after photobleaching, which was decreased by the gap junction blocker carbenoxolone (CBX). In these cultures, we determined the cell type-specific effects of CBX on the excitotoxic damage caused by N-methyl-D-aspartate (NMDA). We determined in both astrocytes and neurons the influence of CBX, alone or together with NMDA challenge, on cytotoxicity using propidium iodide labeling. CBX alone was not cytotoxic, but CBX treatment differentially accelerated the NMDA-induced cell death in both astrocytes and neurons. In addition, we measured mitochondrial potential using rhodamine 123, membrane potential using the oxonol dye bis(1,3-diethylthiobarbituric acid)trimethine oxonol, cytosolic Ca(2+) level using fura-2, and formation of reactive oxygen species (ROS) using dihydroethidium. CBX alone induced neither an intracellular Ca(2+) rise nor a membrane depolarization. However, CBX elicited a mitochondrial depolarization in both astrocytes and neurons and increased the ROS formation in neurons. In contrast, NMDA caused a membrane depolarization in neurons, coinciding with intracellular Ca(2+) rise, but neither mitochondrial depolarization nor ROS production seem to be involved in NMDA-mediated cytotoxicity. Pre-treatment with CBX accelerated the NMDA-induced membrane depolarization and prevented the repolarization of neurons after the NMDA challenge. We hypothesize that these effects are possibly mediated via blockage of gap junctions, and might be involved in the mechanism of CBX-induced acceleration of excitotoxic cell death, whereas the CBX-induced mitochondrial depolarization and ROS formation are not responsible for the increase in cytotoxicity. We conclude that

  7. Specificity of gap junction communication among human mammary cells and connexin transfectants in culture

    PubMed Central

    1993-01-01

    In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC

  8. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

    PubMed Central

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A.; Dilly, Suzanne J.; James, Sean; Dimitri, Wade; Ladwa, Sweta R.; Taylor, Paul C.; Singer, Donald R. J.

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  9. [Effects of Chinese herbal compound for supplementing qi and activating blood circulation on actin, Cx43 expressions and gap junctional intercellular communication functions of myocardial cells in patients with Coxsackie virus B 3 viral myocarditis].

    PubMed

    Zhang, Ming-xue; He, Wei; Gu, Ping

    2010-08-01

    To observe the effect of Chinese herbal compound for supplementing qi and activating blood circulation (CHC) on the gap junctional intercellular communication (GJIC) function of myocardial cells in patients with Coxsackie virus B 3 (CVB3) viral myocarditis. Expressions of actin and connexin43 (Cx43) in myocardial cells of patients arranged in three groups (the normal control group, the viral infected group and the CHC treated group) were detected by immunohistochemical method; the fluorescence photobleaching recovery rate of cells was detected by laser scanning confocal microscope. As compared with the viral infected group, the expressions of actin and Cx43 were increased and the GJIC function was improved in the CHC treated group. CHC could antagonize viral injury on skeleton protein, and repair the structure of gap junction channel to improve the GJIC function of myocardial cells after being attacked by CVB3.

  10. Inhibition of the gap junctional component of endothelium-dependent relaxations in rabbit iliac artery by 18-α glycyrrhetinic acid

    PubMed Central

    Taylor, Hannah J; Chaytor, Andrew T; Evans, W Howard; Griffith, Tudor M

    1998-01-01

    The gap junction inhibitor 18-α-glycyrrhetinic acid (α-GA, 100 μM) attenuated endothelium-dependent relaxations to acetylcholine and cyclopiazonic acid by ∼20% in rings of pre-constricted rabbit iliac artery. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 300 μM) inhibited relaxations to both agents by ∼65% and these were further attenuated by α-GA to <10% of control. In endothelium-denuded preparations, relaxations to sodium nitroprusside were not affected by α-GA. Heterocellular gap junctional communication may therefore account for nitric oxide-independent relaxations evoked both by receptor-dependent and -independent mechanisms in rabbit iliac artery. PMID:9776336

  11. Transient suppression of gap junctional intercellular communication after exposure to 100-nanosecond pulsed electric fields.

    PubMed

    Steuer, Anna; Schmidt, Anke; Labohá, Petra; Babica, Pavel; Kolb, Juergen F

    2016-12-01

    Gap junctional intercellular communication (GJIC) is an important mechanism that is involved and affected in many diseases and injuries. So far, the effect of nanosecond pulsed electric fields (nsPEFs) on the communication between cells was not investigated. An in vitro approach is presented with rat liver epithelial WB-F344 cells grown and exposed in a monolayer. In order to observe sub-lethal effects, cells were exposed to pulsed electric fields with a duration of 100ns and amplitudes between 10 and 20kV/cm. GJIC strongly decreased within 15min after treatment but recovered within 24h. Gene expression of Cx43 was significantly decreased and associated with a reduced total amount of Cx43 protein. In addition, MAP kinases p38 and Erk1/2, involved in Cx43 phosphorylation, were activated and Cx43 became hyperphosphorylated. Immunofluorescent staining of Cx43 displayed the disassembly of gap junctions. Further, a reorganization of the actin cytoskeleton was observed whereas tight junction protein ZO-1 was not significantly affected. All effects were field- and time-dependent and most pronounced within 30 to 60min after treatment. A better understanding of a possible manipulation of GJIC by nsPEFs might eventually offer a possibility to develop and improve treatments. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Effects of geometric configuration on droplet generation in Y-junctions and anti-Y-junctions microchannels

    NASA Astrophysics Data System (ADS)

    Liu, Zhao-Miao; Liu, Li-Kun; Shen, Feng

    2015-10-01

    Droplets generation in Y-junctions and anti-Y-junctions microchannels are experimentally studied using a high speed digital microscopic system and numerical simulation. Geometric configuration of a microchannel, such as Y-angle (90°, 135°, -90° and -135°), channel depth and other factors have been taken into consideration. It is found that droplets generated in anti-Y-junctions have a smaller size and a shorter generation cycle compared with those in Y-junctions under the same experimental conditions. Through observing the internal velocity field, the vortex appearing in continuous phase in anti-Y-junctions is one of the key factors for the difference of droplet size and generation cycle. It is found that droplet size is bigger and generation cycle is longer when the absolute angle value of the intersection between the continuous and the dispersed phases (i.e., the angle between the main channel and the continuous phase or the dispersed phase channel) increases. The droplet's size is influenced by the Y-angle, which varies with the channel depth in Y-junctions. The Y-angle has a positive effect on the droplet generation cycle, but a smaller height-width ratio will enhance the impact of a continuous and dispersed phase's intersection angle on the droplet generation cycle in Y-junctions microchannels.

  13. Gap Junction Proteins in the Blood-Brain Barrier Control Nutrient-Dependent Reactivation of Drosophila Neural Stem Cells

    PubMed Central

    Spéder, Pauline; Brand, Andrea H.

    2014-01-01

    Summary Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion. PMID:25065772

  14. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less

  15. Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

    PubMed

    Corteggio, A; Florio, J; Roperto, F; Borzacchiello, G

    2011-01-01

    The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. An intercellular polyamine transfer via gap junctions regulates proliferation and response to stress in epithelial cells

    PubMed Central

    Desforges, Bénédicte; Curmi, Patrick A.; Bounedjah, Ouissame; Nakib, Samir; Hamon, Loic; De Bandt, Jean-Pascal; Pastré, David

    2013-01-01

    In the organism, quiescent epithelial cells have the potential to resume cycling as a result of various stimuli, including wound healing or oxidative stress. Because quiescent cells have a low polyamine level, resuming their growth requires an increase of their intracellular polyamine levels via de novo polyamine synthesis or their uptake from plasma. Another alternative, explored here, is an intercellular exchange with polyamine-rich cycling cells via gap junctions. We show that polyamines promote gap junction communication between proliferating cells by promoting dynamical microtubule plus ends at the cell periphery and thus allow polyamine exchange between cells. In this way, cycling cells favor regrowth in adjacent cells deprived of polyamines. In addition, intercellular interactions mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration. PMID:23515223

  17. Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions.

    PubMed

    Toranzo, G Sánchez; Oterino, J; Zelarayán, L; Bonilla, F; Bühler, M I

    2007-02-01

    It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as

  18. Bridging the gap to therapeutic strategies based on connexin/pannexin biology.

    PubMed

    Naus, Christian C; Giaume, Christian

    2016-11-29

    A unique workshop was recently held focusing on enhancing collaborations leading to identify and update the development of therapeutic strategies targeting connexin/pannexin large pore channels. Basic scientists exploring the functions of these channels in various pathologies gathered together with leading pharma companies which are targeting gap junction proteins for specific therapeutic applications. This highlights how paths of discovery research can converge with therapeutic strategies in innovative ways to enhance target identification and validation.

  19. Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9.

    PubMed

    Boucherie, Sylviane; Decaens, Catherine; Verbavatz, Jean-Marc; Grosse, Brigitte; Erard, Marie; Merola, Fabienne; Cassio, Doris; Combettes, Laurent

    2013-12-01

    Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity. We showed that the toxicity of CdCl2 (1 µM, 24 h) resulted in a reduction in direct intercellular communication (via gap junctions) and in an increase in paracellular permeability (decrease in the sealing of tight junctions). These effects were not related to changes in the expression of the key proteins involved, Cx32 and claudin 2, the first being constitutive of gap junctions and the second of tight junctions in this cell line. Using immunofluorescence experiments, we observed a change in the location of Cx32 and claudin 2: these two proteins were less often found in the tight junction network that closes the bile canaliculi (BC). In control cells, 'Proximity Ligation Assay' (PLA Duolink®) has confirmed in situ that molecules of claudin 2 and Cx32 are very close to each other at the BC (probably less than 16 nm). This was no longer the case after treatment with CdCl2 . Localisation of occludin and Cx32 relative to each other was not modified by CdCl2 , but CdCl2 increased the PLA signal between molecules of JAM-A and Cx32. Finally, examination of freeze-fracture replicas obtained from cultures treated with CdCl2 showed the disruption of the network of tight junctions and the depletion or the disintegration of the junctional plaques associated with tight junctions. This study demonstrates in situ the changes induced by cadmium on the organisation of cell-cell junctions and points out the importance of the association Cx32/claudin 2 for the maintenance of normal hepatocyte functions. © 2013 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  20. p - n Junction Dynamics Induced in a Graphene Channel by Ferroelectric-Domain Motion in the Substrate

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kurchak, Anatolii I.; Eliseev, Eugene A.; Kalinin, Sergei V.

    The p - n junction dynamics induced in a graphene channel by stripe-domain nucleation, motion, and reversal in a ferroelectric substrate is explored using a self-consistent approach based on Landau-Ginzburg-Devonshire phenomenology combined with classical electrostatics. Relatively low gate voltages are required to induce the hysteresis of ferroelectric polarization and graphene charge in response to the periodic gate voltage. Pronounced nonlinear hysteresis of graphene conductance with a wide memory window corresponds to high amplitudes of gate voltage. Also, we reveal the extrinsic size effect in the dependence of the graphene-channel conductivity on its length. We predict that the top-gate–dielectric-layer–graphene-channel–ferroelectric-substrate nanostructure consideredmore » here can be a promising candidate for the fabrication of the next generation of modulators and rectifiers based on the graphene p - n junctions.« less

  1. Increase in gap junctional intercellular communication by high molecular weight hyaluronic acid associated with fibroblast growth factor 2 and keratinocyte growth factor production in normal human dermal fibroblasts.

    PubMed

    Park, Jeong Ung; Tsuchiya, Toshie

    2002-07-01

    The effects of different molecular weights of hyaluronic acid (HA), a major component of extracellular matrix, on gap junctional intercellular communication (GJIC) in normal human dermal fibroblasts (NHDF cells) were investigated. NHDF cells were cultured for 4 days with different molecular weights of HA and then the extent of GJIC was assessed by the scrape-loading dye transfer method, using Lucifer yellow. The area of dye transfer was greater in the dishes coated with HA than in those to which HA was added. Thus, NHDF cells cultured on surfaces coated with high molecular weight (HMW) HA (MW, 800 kDa) showed greatly enhanced GJIC. Furthermore, another aim of this study was to evaluate the effects of different molecular weights of HA on the production of FGF-2 and KGF, because both are important cytokines produced by NHDF cells. When FGF-2 and KGF cultured levels of cell extracts and media were determined by ELISA, both levels were significantly enhanced when cells were grown on plates coated with HMW HA. This finding indicated that the function of gap junction channels in NHDF cells grown on plates coated with HMW HA may promote the biosynthesis of growth factors such as FGF-2 and KGF.

  2. Interaction between fractional Josephson vortices in multi-gap superconductor tunnel junctions

    NASA Astrophysics Data System (ADS)

    Kim, Ju H.

    In a long Josephson junction (LJJ) with two-band superconductors, fractionalization of Josephson vortices (fluxons) can occur in the broken time reversal symmetry state when spatial phase textures (i-solitons) are excited. Excitation of i-solitons in each superconductor layer of the junction, arising due to the presence of two condensates and the interband Josephson effect, leads to spatial variation of the critical current density between the superconductor layers. Similar to the situation in a YBa2 Cu3O7 - x superconductor film grain boundary, this spatial dependence of the crtitical current density can self-generate magnetic flux in the insulator layer, resulting in fractional fluxons with large and small fraction of flux quantum. Similar to fluxons in one-band superconductor LJJ, these fractional fluxons are found to interact with each other. The interaction between large and small fractional fluxons determines the size of a fluxon which includes two (one large and one small) fractional fluxons. We discuss the nature of interaction between fractional fluxons and suggest that i-soliton excitations in multi-gap superconductor LJJs may be probed by using magnetic flux measurements.

  3. Connexin 43 and ATP-sensitive potassium channels crosstalk: a missing link in hypoxia/ischemia stress.

    PubMed

    Ahmad Waza, Ajaz; Ahmad Bhat, Shabir; Ul Hussain, Mahboob; Ganai, Bashir A

    2018-02-01

    Connexin 43 (Cx43) is a gap junction protein expressed in various tissues and organs of vertebrates. Besides functioning as a gap junction, Cx43 also regulates diverse cellular processes like cell growth and differentiation, cell migration, cell survival, etc. Cx43 is critical for normal cardiac functioning and is therefore abundantly expressed in cardiomyocytes. On the other hand, ATP-sensitive potassium (K ATP ) channels are metabolic sensors converting metabolic changes into electrical activity. These channels are important in maintaining the neurotransmitter release, smooth muscle relaxation, cardiac action potential repolarization, normal physiology of cellular repolarization, insulin secretion and immune function. Cx43 and K ATP channels are part of the same signaling pathway, regulating cell survival during stress conditions and ischemia/hypoxia preconditioning. However, the underlying molecular mechanism for their combined role in ischemia/hypoxia preconditioning is largely unknown. The current review focuses on understanding the molecular mechanism responsible for the coordinated role of Cx43 and K ATP channel protein in protecting cardiomyocytes against ischemia/hypoxia stress.

  4. Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Talhouk, Rabih S.; Mroue, Rana; Mokalled, Mayssa

    2008-11-01

    Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complexmore » components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.« less

  5. Three-junction solar cell

    DOEpatents

    Ludowise, Michael J.

    1986-01-01

    A photovoltaic solar cell is formed in a monolithic semiconductor. The cell contains three junctions. In sequence from the light-entering face, the junctions have a high, a medium, and a low energy gap. The lower junctions are connected in series by one or more metallic members connecting the top of the lower junction through apertures to the bottom of the middle junction. The upper junction is connected in voltage opposition to the lower and middle junctions by second metallic electrodes deposited in holes 60 through the upper junction. The second electrodes are connected to an external terminal.

  6. Role of gap junctions on synchronization in human neocortical networks.

    PubMed

    Gigout, S; Deisz, R A; Dehnicke, C; Turak, B; Devaux, B; Pumain, R; Louvel, J

    2016-04-15

    Gap junctions (GJ) have been implicated in the synchronization of epileptiform activities induced by 4-aminopyrine (4AP) in slices from human epileptogenic cortex. Previous evidence implicated glial GJ to govern the frequency of these epileptiform events. The synchrony of these events (evaluated by the phase unlocking index, PUI) in adjacent areas however was attributed to neuronal GJ. In the present study, we have investigated the effects of GAP-134, a recently developed specific activator of glial GJ, on both the PUI and the frequency of the 4AP-induced epileptiform activities in human neocortical slices of temporal lobe epilepsy tissue. To delineate the impact of GJ on spatial spread of synchronous activity we evaluated the effects of carbenoxolone (CBX, a non-selective GJ blocker) on the spread in three axes 1. vertically in a given cortical column, 2. laterally within the deep cortical layers and 3. laterally within the upper cortical layers. GAP-134 slightly increased the frequency of the 4AP-induced spontaneous epileptiform activities while leaving the PUI unaffected. CBX had no effect on the PUI within a cortical column or on the PUI in the deep cortical layers. CBX increased the PUI for long interelectrodes distances in the upper cortical layers. In conclusion we provide new arguments toward the role played by glial GJ to maintain the frequency of spontaneous activities. We show that neuronal GJ control the PUI only in upper cortical layers. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Innexin-3 forms connexin-like intercellular channels.

    PubMed

    Landesman, Y; White, T W; Starich, T A; Shaw, J E; Goodenough, D A; Paul, D L

    1999-07-01

    Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels.

  8. Complexity of gap junctions between horizontal cells of the carp retina.

    PubMed

    Greb, H; Hermann, S; Dirks, P; Ommen, G; Kretschmer, V; Schultz, K; Zoidl, G; Weiler, R; Janssen-Bienhold, U

    2017-01-06

    In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  9. The role of gap junctions in megakaryocyte-mediated osteoblast proliferation and differentiation.

    PubMed

    Ciovacco, Wendy A; Goldberg, Carolyn G; Taylor, Amanda F; Lemieux, Justin M; Horowitz, Mark C; Donahue, Henry J; Kacena, Melissa A

    2009-01-01

    Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether this response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18alpha-glycyrrhetinic acid (alphaGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MKs to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation.

  10. Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

    PubMed

    Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

    2015-04-01

    The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

  11. Gap junction communication between uterine stromal cells plays a critical role in pregnancy-associated neovascularization and embryo survival.

    PubMed

    Laws, Mary J; Taylor, Robert N; Sidell, Neil; DeMayo, Francesco J; Lydon, John P; Gutstein, David E; Bagchi, Milan K; Bagchi, Indrani C

    2008-08-01

    In the uterus, the formation of new maternal blood vessels in the stromal compartment at the time of embryonic implantation is critical for the establishment and maintenance of pregnancy. Although uterine angiogenesis is known to be influenced by the steroid hormones estrogen (E) and progesterone (P), the underlying molecular pathways remain poorly understood. Here, we report that the expression of connexin 43 (Cx43), a major gap junction protein, is markedly enhanced in response to E in uterine stromal cells surrounding the implanted embryo during the early phases of pregnancy. Conditional deletion of the Cx43 gene in these stromal cells and the consequent disruption of their gap junctions led to a striking impairment in the development of new blood vessels within the stromal compartment, resulting in the arrest of embryo growth and early pregnancy loss. Further analysis of this phenotypical defect revealed that loss of Cx43 expression resulted in aberrant differentiation of uterine stromal cells and impaired production of several key angiogenic factors, including the vascular endothelial growth factor (Vegf). Ablation of CX43 expression in human endometrial stromal cells in vitro led to similar findings. Collectively, these results uncovered a unique link between steroid hormone-regulated cell-cell communication within the pregnant uterus and the development of an elaborate vascular network that supports embryonic growth. Our study presents the first evidence that Cx43-type gap junctions play a critical and conserved role in modulating stromal differentiation, and regulate the consequent production of crucial paracrine signals that control uterine neovascularization during implantation.

  12. Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

    PubMed

    Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

    2014-04-01

    Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. Copyright © 2014 Wiley Periodicals, Inc.

  13. On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells

    PubMed Central

    Vargas, Anibal A.; Cisterna, Bruno A.; Saavedra-Leiva, Fujiko; Urrutia, Carolina; Cea, Luis A.; Vielma, Alex H.; Gutierrez-Maldonado, Sebastian E.; Martin, Alberto J. M.; Pareja-Barrueto, Claudia; Escalona, Yerko; Schmachtenberg, Oliver; Lagos, Carlos F.; Perez-Acle, Tomas; Sáez, Juan C.

    2017-01-01

    Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs

  14. Enhanced Andreev reflection in gapped graphene

    NASA Astrophysics Data System (ADS)

    Majidi, Leyla; Zareyan, Malek

    2012-08-01

    We theoretically demonstrate unusual features of superconducting proximity effect in gapped graphene that presents a pseudospin symmetry-broken ferromagnet with a net pseudomagnetization. We find that the presence of a band gap makes the Andreev conductance of graphene superconductor/pseudoferromagnet (S/PF) junction to behave similar to that of a graphene ferromagnet-superconductor junction. The energy gap ΔN can enhance the pseudospin inverted Andreev conductance of S/PF junction to reach a limiting maximum value for ΔN≫μ, which depending on the bias voltage can be larger than the value for the corresponding junction with no energy gap. We further demonstrate a damped-oscillatory behavior for the local density of states of the PF region of S/PF junction and a long-range crossed Andreev reflection process in PF/S/PF structure with antiparallel alignment of pseudomagnetizations of PFs, which confirm that, in this respect, the gapped normal graphene behaves like a ferromagnetic graphene.

  15. Gap features of layered iron-selenium-tellurium compound below and above the superconducting transition temperature by break-junction spectroscopy combined with STS

    NASA Astrophysics Data System (ADS)

    Ekino, T.; Sugimoto, A.; Gabovich, A. M.

    2018-05-01

    We studied correlations between the superconducting gap features of Te-substituted FeSe observed by scanning tunnelling spectroscopy (STS) and break-junction tunnelling spectroscopy (BJTS). At bias voltages outside the superconducting gap-energy range, the broad gap structure exists, which becomes the normal-state gap above the critical temperature, T c. Such behaviour is consistent with the model of the partially gapped density-wave superconductor involving both superconducting gaps and pseudogaps, which has been applied by us earlier to high-Tc cuprates. The similarity suggests that the parent electronic spectrum features should have much in common for these classes of materials.

  16. Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

    PubMed

    Rivedal, Edgar; Leithe, Edward

    2005-01-15

    The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

  17. [The effect of 18beta-glycyrrhetinic acid on gap junction among cerebral arteriolar smooth muscle cells in Wistar rat and spontaneously hypertensive rat].

    PubMed

    Chen, Xin-Yan; Si, Jun-Qiang; Li, Li; Zhao, Lei; Wei, Li-Li; Jiang, Xue-Wei; Ma, Ke-Tao

    2013-05-01

    This study compared Wistar rat with spontaneously hypertensive rat (SHR) on the electrophysiology and coupling force of the smooth muscle cells in the cerebral arteriolar segments and observe the influence of 18beta-glycyrrhetinic acid(18beta-GA) on the gap junctions between the arterial smooth muscle cells. The outer layer's connective tissue of the cerebral arteriolar segments was removed. Whole-cell patch clamp recordings were used to observe the 18beta-GA's impaction on the arteriolar segment membrane's input capacitance (C(input)), input conductance (G(input)) and input resistance (R(input)) of the smooth muscle cells. (1) The C(input) and G(input) of the SHR arteriolar segment smooth muscle cells was much higher than the Wistar rats, there was significant difference (P < 0.05). (2) 18beta-GA concentration-dependently reduced C(input) and G(input) (or increase R(input)) on smooth muscle cells in arteriolar segment. IC50 of 18beta-GA suppression's G(input) of the Wistar rat and SHR were 1.7 and 2.0 micromol/L respectively, there was not significant difference (P > 0.05). After application of 18beta-GA concentration > or = 100 micrmol/L, the C(input), G(input) and R(input) of the single smooth muscle cells was very close. Gap junctional coupling is enhanced in the SHR cerebral arterial smooth muscle cells. 18beta-GA concentration-dependent inhibits Wistar rat's and SHR cerebral arteriolar gap junctions between arterial smooth muscle cells. The inhibitory potency is similar between the two different rats. When 18beta-GA concentration is > or = 100 micromol/L, it can completely block gap junctions between arteriolar smooth muscle cells.

  18. Connexin Channel Permeability to Cytoplasmic Molecules

    PubMed Central

    Harris, Andrew L.

    2007-01-01

    Connexin channels are known to be permeable to a variety of cytoplasmic molecules. The first observation of second messenger junctional permeability, made ∼30 years ago, sparked broad interest in gap junction channels as mediators of intercellular molecular signaling. Since then, much has been learned about the diversity of connexin channels with regard to isoform diversity, tissue and developmental distribution, modes of channel regulation, assembly and expression, biochemical modification and permeability, all of which appear to be dynamically regulated. This information has expanded the potential roles of connexin channels in development, physiology and disease, and made their elucidation much more complex - 30 years ago such an orchestra of junctional dynamics was unanticipated. Only recently, however, have investigators been able to directly address, in this more complex framework, the key issue: What specific biological molecules, second messengers and others, are able to permeate the various types of connexin channels, and how well? An important related issue, given the ever-growing list of connexin-related pathologies, is how these permeabilities are altered by disease-causing connexin mutations. Together, many studies show that a variety of cytoplasmic molecules can permeate the different types of connexin channels. A few studies reveal differences in permeation by different molecules through a particular type of connexin channel, and differences in permeation by a particular molecule through different types of connexin channels. This article describes and evaluates the various methods used to obtain these data, presents an annotated compilation of the results, and discusses the findings in the context of what can be inferred about mechanism of selectivity and potential relevance to signaling. The data strongly suggest that highly specific interactions take place between connexin pores and specific biological molecular permeants, and that those

  19. The Role of Gap Junctions in Megakaryocyte-Mediated Osteoblast Proliferation and Differentiation

    PubMed Central

    Ciovacco, Wendy A.; Goldberg, Carolyn G.; Taylor, Amanda F.; Lemieux, Justin M.; Horowitz, Mark C.; Donahue, Henry J.; Kacena, Melissa A.

    2009-01-01

    Gap junctions (GJs) are membrane-spanning channels that facilitate intercellular communication by allowing small signaling molecules (e.g. calcium ions, inositol phosphates, and cyclic nucleotides) to pass from cell to cell. Over the past two decades, many studies have described a role for GJ intercellular communication (GJIC) in the proliferation and differentiation of many cells, including bone cells. Recently, we reported that megakaryocytes (MKs) enhance osteoblast (OB) proliferation by a juxtacrine signaling mechanism. Here we determine whether that response is facilitated by GJIC. First we demonstrate that MKs express connexin 43 (Cx43), the predominant GJ protein expressed by bone cells, including OBs. Next, we provide data showing that MKs can communicate with OBs via GJIC, and that the addition of two distinct GJ uncouplers, 18α-glycyrrhetinic acid (αGA) or oleamide, inhibits this communication. We then demonstrate that inhibiting MK-mediated GJIC further enhances the ability of MK to stimulate OB proliferation. Finally, we show that while culturing MKs with OBs reduces gene expression of several differentiation markers/matrix proteins (type I collagen, osteocalcin, and alkaline phosphatase), reduces alkaline phosphatase enzymatic activity, and decreases mineralization in OBs, blocking GJIC does not result in MK-induced reductions in OB gene expression, enzymatic levels, or mineralized nodule formation. Overall, these data provide evidence that GJIC between MKs and OBs is functional, and that inhibiting GJIC in MK-OB cultures enhances OB proliferation without apparently altering differentiation when compared to similarly treated OB cultures. Thus, these observations regarding MK-OB GJIC inhibition may provide insight regarding potential novel targets for anabolic bone formation. PMID:18848655

  20. BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice.

    PubMed

    Crispino, Giulia; Di Pasquale, Giovanni; Scimemi, Pietro; Rodriguez, Laura; Galindo Ramirez, Fabian; De Siati, Romolo Daniele; Santarelli, Rosa Maria; Arslan, Edoardo; Bortolozzi, Mario; Chiorini, John A; Mammano, Fabio

    2011-01-01

    The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

  1. Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

    PubMed Central

    Liu, Xijuan; Wu, Yingya; Du, Biaoyan; Li, Jiefen; Zhou, Jing; Li, Jingjing; Tan, Yuhui

    2013-01-01

    The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy. PMID:23861780

  2. Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.

    PubMed

    Lacar, Benjamin; Young, Stephanie Z; Platel, Jean-Claude; Bordey, Angélique

    2011-12-01

    In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  3. Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission.

    PubMed

    Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A

    2013-06-15

    Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction 'buds' suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior.

  4. Visualizing the effect of dynamin inhibition on annular gap vesicle formation and fission

    PubMed Central

    Nickel, Beth; Boller, Marie; Schneider, Kimberly; Shakespeare, Teresa; Gay, Vernon; Murray, Sandra A.

    2013-01-01

    Summary Although gap junction plaque assembly has been extensively studied, mechanisms involved in plaque disassembly are not well understood. Disassembly involves an internalization process in which annular gap junction vesicles are formed. These vesicles undergo fission, but the molecular machinery needed for these fissions has not been described. The mechanoenzyme dynamin has been previously demonstrated to play a role in gap junction plaque internalization. To investigate the role of dynamin in annular gap junction vesicle fission, immunocytochemical, time-lapse and transmission electron microscopy were used to analyze SW-13 adrenocortical cells in culture. Dynamin was demonstrated to colocalize with gap junction plaques and vesicles. Dynamin inhibition, by siRNA knockdown or treatment with the dynamin GTPase inhibitor dynasore, increased the number and size of gap junction ‘buds’ suspended from the gap junction plaques. Buds, in control populations, were frequently released to form annular gap junction vesicles. In dynamin-inhibited populations, the buds were larger and infrequently released and thus fewer annular gap junction vesicles were formed. In addition, the number of annular gap junction vesicle fissions per hour was reduced in the dynamin-inhibited populations. We believe this to be the first report addressing the details of annular gap junction vesicle fissions and demonstrating a role of dynamin in this process. This information is crucial for elucidating the relationship between gap junctions, membrane regulation and cell behavior. PMID:23591819

  5. Equal-Spin Andreev Reflection on Junctions of Spin-Resolved Quantum Hall Bulk State and Spin-Singlet Superconductor.

    PubMed

    Matsuo, Sadashige; Ueda, Kento; Baba, Shoji; Kamata, Hiroshi; Tateno, Mizuki; Shabani, Javad; Palmstrøm, Christopher J; Tarucha, Seigo

    2018-02-22

    The recent development of superconducting spintronics has revealed the spin-triplet superconducting proximity effect from a spin-singlet superconductor into a spin-polarized normal metal. In addition recently superconducting junctions using semiconductors are in demand for highly controlled experiments to engineer topological superconductivity. Here we report experimental observation of Andreev reflection in junctions of spin-resolved quantum Hall (QH) states in an InAs quantum well and the spin-singlet superconductor NbTi. The measured conductance indicates a sub-gap feature and two peaks on the outer side of the sub-gap feature in the QH plateau-transition regime increases. The observed structures can be explained by considering transport with Andreev reflection from two channels, one originating from equal-spin Andreev reflection intermediated by spin-flip processes and second arising from normal Andreev reflection. This result indicates the possibility to induce the superconducting proximity gap in the the QH bulk state, and the possibility for the development of superconducting spintronics in semiconductor devices.

  6. Ion Channels in Brain Metastasis

    PubMed Central

    Klumpp, Lukas; Sezgin, Efe C.; Eckert, Franziska; Huber, Stephan M.

    2016-01-01

    Breast cancer, lung cancer and melanoma exhibit a high metastatic tropism to the brain. Development of brain metastases severely worsens the prognosis of cancer patients and constrains curative treatment options. Metastasizing to the brain by cancer cells can be dissected in consecutive processes including epithelial–mesenchymal transition, evasion from the primary tumor, intravasation and circulation in the blood, extravasation across the blood–brain barrier, formation of metastatic niches, and colonization in the brain. Ion channels have been demonstrated to be aberrantly expressed in tumor cells where they regulate neoplastic transformation, malignant progression or therapy resistance. Moreover, many ion channel modulators are FDA-approved drugs and in clinical use proposing ion channels as druggable targets for future anti-cancer therapy. The present review article aims to summarize the current knowledge on the function of ion channels in the different processes of brain metastasis. The data suggest that certain channel types involving voltage-gated sodium channels, ATP-release channels, ionotropic neurotransmitter receptors and gap junction-generating connexins interfere with distinct processes of brain metastazation. PMID:27618016

  7. Ge p-channel tunneling FETs with steep phosphorus profile source junctions

    NASA Astrophysics Data System (ADS)

    Takaguchi, Ryotaro; Matsumura, Ryo; Katoh, Takumi; Takenaka, Mitsuru; Takagi, Shinichi

    2018-04-01

    The solid-phase diffusion processes of three n-type dopants, i.e., phosphorus (P), arsenic (As), and antimony (Sb), from spin-on-glass (SOG) into Ge are compared. We show that P diffusion can realize both the highest impurity concentration (˜7 × 1019 cm-3) and the steepest impurity profile (˜10 nm/dec) among the cases of the three n-type dopants because the diffusion coefficient is strongly dependent on the dopant concentration. As a result, we can conclude that P is the most suitable dopant for the source formation of Ge p-channel TFETs. Using this P diffusion, we fabricate Ge p-channel TFETs with high-P-concentration and steep-P-profile source junctions and demonstrate their operation. A high ON current of ˜1.7 µA/µm is obtained at room temperature. However, the subthreshold swing and ON current/OFF current ratio are degraded by any generation-recombination-related current component. At 150 K, SSmin of ˜108 mV/dec and ON/OFF ratio of ˜3.5 × 105 are obtained.

  8. Distinctive fingerprints of erosional regimes in terrestrial channel networks

    NASA Astrophysics Data System (ADS)

    Grau Galofre, A.; Jellinek, M.

    2017-12-01

    Satellite imagery and digital elevation maps capture the large scale morphology of channel networks attributed to long term erosional processes, such as fluvial, glacial, groundwater sapping and subglacial erosion. Characteristic morphologies associated with each of these styles of erosion have been studied in detail, but there exists a knowledge gap related to their parameterization and quantification. This knowledge gap prevents a rigorous analysis of the dominant processes that shaped a particular landscape, and a comparison across styles of erosion. To address this gap, we use previous morphological descriptions of glaciers, rivers, sapping valleys and tunnel valleys to identify and measure quantitative metrics diagnostic of these distinctive styles of erosion. From digital elevation models, we identify four geometric metrics: The minimum channel width, channel aspect ratio (longest length to channel width at the outlet), presence of undulating longitudinal profiles, and tributary junction angle. We also parameterize channel network complexity in terms of its stream order and fractal dimension. We then perform a statistical classification of the channel networks using a Principal Component Analysis on measurements of these six metrics on a dataset of 70 channelized systems. We show that rivers, glaciers, groundwater seepage and subglacial meltwater erode the landscape in rigorously distinguishable ways. Our methodology can more generally be applied to identify the contributions of different processes involved in carving a channel network. In particular, we are able to identify transitions from fluvial to glaciated landscapes or vice-versa.

  9. Mechanics of Fluid-Filled Interstitial Gaps. II. Gap Characteristics in Xenopus Embryonic Ectoderm.

    PubMed

    Barua, Debanjan; Parent, Serge E; Winklbauer, Rudolf

    2017-08-22

    The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1-4 μm side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m 2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Neuroprotective Role of Gap Junctions in a Neuron Astrocyte Network Model.

    PubMed

    Huguet, Gemma; Joglekar, Anoushka; Messi, Leopold Matamba; Buckalew, Richard; Wong, Sarah; Terman, David

    2016-07-26

    A detailed biophysical model for a neuron/astrocyte network is developed to explore mechanisms responsible for the initiation and propagation of cortical spreading depolarizations and the role of astrocytes in maintaining ion homeostasis, thereby preventing these pathological waves. Simulations of the model illustrate how properties of spreading depolarizations, such as wave speed and duration of depolarization, depend on several factors, including the neuron and astrocyte Na(+)-K(+) ATPase pump strengths. In particular, we consider the neuroprotective role of astrocyte gap junction coupling. The model demonstrates that a syncytium of electrically coupled astrocytes can maintain a physiological membrane potential in the presence of an elevated extracellular K(+) concentration and efficiently distribute the excess K(+) across the syncytium. This provides an effective neuroprotective mechanism for delaying or preventing the initiation of spreading depolarizations. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. Direct antigen presentation and gap junction mediated cross-presentation during apoptosis.

    PubMed

    Pang, Baoxu; Neijssen, Joost; Qiao, Xiaohang; Janssen, Lennert; Janssen, Hans; Lippuner, Christoph; Neefjes, Jacques

    2009-07-15

    MHC class I molecules present peptides from endogenous proteins. Ags can also be presented when derived from extracellular sources in the form of apoptotic bodies. Cross-presentation of such Ags by dendritic cells is required for proper CTL responses. The fate of Ags in cells initiated for apoptosis is unclear as is the mechanism of apoptosis-derived Ag transfer into dendritic cells. Here we show that novel Ags can be generated by caspases and be presented by MHC class I molecules of apoptotic cells. Since gap junctions function until apoptotic cells remodel to form apoptotic bodies, transfer and cross-presentation of apoptotic peptides by neighboring and dendritic cells occurs. We thus define a novel phase in classical Ag presentation and cross-presentation by MHC class I molecules: presentation of Ags created by caspase activities in cells in apoptosis.

  12. Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.

    PubMed

    Mayan, Maria D; Gago-Fuentes, Raquel; Carpintero-Fernandez, Paula; Fernandez-Puente, Patricia; Filgueira-Fernandez, Purificacion; Goyanes, Noa; Valiunas, Virginijus; Brink, Peter R; Goldberg, Gary S; Blanco, Francisco J

    2015-01-01

    This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue. Published by the BMJ Publishing Group Limited. For

  13. Tunnel junction multiple wavelength light-emitting diodes

    DOEpatents

    Olson, Jerry M.; Kurtz, Sarah R.

    1992-01-01

    A multiple wavelength LED having a monolithic cascade cell structure comprising at least two p-n junctions, wherein each of said at least two p-n junctions have substantially different band gaps, and electrical connector means by which said at least two p-n junctions may be collectively energized; and wherein said diode comprises a tunnel junction or interconnect.

  14. Differential involvement of ipsilateral and contralateral spinal cord astrocyte D-serine in carrageenan-induced mirror-image pain: role of σ1 receptors and astrocyte gap junctions.

    PubMed

    Choi, Hoon-Seong; Roh, Dae-Hyun; Yoon, Seo-Yeon; Choi, Sheu-Ran; Kwon, Soon-Gu; Kang, Suk-Yun; Moon, Ji-Young; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2018-02-01

    Although we have recently demonstrated that spinal astrocyte gap junctions mediate the development of mirror-image pain (MIP), it is still unclear which astrocyte-derived factor is responsible for the development of MIP and how its production is controlled. In the present study, we focused on the role of ipsilateral versus contralateral D-serine in the development of MIP and investigated the possible involvement of σ1 receptors and gap junctions in astrocyte D-serine production. Following carrageenan injection, mechanical allodynia was tested at various time points to examine the effect of individual drugs. Immunohistochemistry and Western blot analyses were performed to clarify the expression levels of spinal D-serine, serine racemase, σ1 receptors and connexin 43. The expression of ipsilateral D-serine was up-regulated during the early phase of inflammation, while contralateral D-serine increased during the later phase of inflammation. The pharmacological inhibition of D-serine during the early phase blocked the development of both ipsilateral and contralateral mechanical allodynia. However, the inhibition of D-serine during the later phase of inflammation blocked contralateral, but not ipsilateral mechanical allodynia. Furthermore, the inhibition of σ1 receptors during the earlier phase of inflammation inhibited the increase in ipsilateral D-serine. Conversely, the blockade of astrocyte gap junctions suppressed the up-regulation of contralateral D-serine during the later phase of inflammation. Spinal astrocyte D-serine plays an important role in the development of mirror-image pain. Furthermore, σ1 receptors and astrocyte gap junction signalling mediate ipsilateral and contralateral D-serine production respectively. © 2017 The British Pharmacological Society.

  15. Connexin channels and phospholipids: association and modulation

    PubMed Central

    Locke, Darren; Harris, Andrew L

    2009-01-01

    Background For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood. Results Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred. Conclusion This study is the first to

  16. STIM proteins and the endoplasmic reticulum-plasma membrane junctions.

    PubMed

    Carrasco, Silvia; Meyer, Tobias

    2011-01-01

    Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.

  17. Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers.

    PubMed

    Samak, Geetha; Gangwar, Ruchika; Meena, Avtar S; Rao, Roshan G; Shukla, Pradeep K; Manda, Bhargavi; Narayanan, Damodaran; Jaggar, Jonathan H; Rao, RadhaKrishna

    2016-12-13

    Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca 2+ -free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or Ca V 1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.

  18. Tunnel junction multiple wavelength light-emitting diodes

    DOEpatents

    Olson, J.M.; Kurtz, S.R.

    1992-11-24

    A multiple wavelength LED having a monolithic cascade cell structure comprising at least two p-n junctions, wherein each of said at least two p-n junctions have substantially different band gaps, and electrical connector means by which said at least two p-n junctions may be collectively energized; and wherein said diode comprises a tunnel junction or interconnect. 5 figs.

  19. Dielectric properties of biological tissues in which cells are connected by communicating junctions

    NASA Astrophysics Data System (ADS)

    Asami, Koji

    2007-06-01

    The frequency dependence of the complex permittivity of biological tissues has been simulated using a simple model that is a cubic array of spherical cells in a parallel plate capacitor. The cells are connected by two types of communicating junctions: one is a membrane-lined channel for plasmodesmata in plant tissues, and the other is a conducting patch of adjoining plasma membranes for gap junctions in animal tissues. Both junctions provided similar effects on the dielectric properties of the tissue model. The model without junction showed a dielectric relaxation (called β-dispersion) that was expected from an interfacial polarization theory for a concentrated suspension of spherical cells. The dielectric relaxation was the same as that of the model in which neighbouring cells were connected by junctions perpendicular to the applied electric field. When neighbouring cells were connected by junctions parallel to the applied electric field or in all directions, a dielectric relaxation appeared at a lower frequency side in addition to the β-dispersion, corresponding to the so called α-dispersion. When junctions were randomly introduced at varied probabilities Pj, the low-frequency (LF) relaxation curve became broader, especially at Pj of 0.2-0.5, and its intensity was proportional to Pj up to 0.7. The intensity and the characteristic frequency of the LF relaxation both decreased with decreasing junction conductance. The simulations indicate that communicating junctions are important for understanding the LF dielectric relaxation in tissues.

  20. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    NASA Astrophysics Data System (ADS)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  1. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

    PubMed

    Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

    2015-04-28

    Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

  2. cAMP enhances Cx43 gap junction formation and function and reverses choline deficiency apoptosis.

    PubMed

    Albright, C D; Kuo, J; Jeong, S

    2001-08-01

    Previously, it had been shown that acute choline deficiency (CD) induced apoptosis in cultured rat liver epithelial cells, whereas cells that are adapted to survive in low-choline-containing medium acquire resistance to CD apoptosis and undergo malignant transformation. Thus, understanding the mechanisms of action of CD could increase our understanding of the role of choline, an essential nutrient, in the process of malignant transformation. The present experiments were designed to test the hypothesis that CD might function as a pro-apoptotic trigger by altering the localization of connexin 43 gap junction protein and gap junctional intercellular communication (GJIC). Established liver epithelial cells (WB cells; Hep3B cells) were maintained in a defined, serum-free medium control (70 microM choline) or choline deficient medium (CD, 5 microM choline) and the localization of connexin 43 protein (Cx43) was studied by immunocytochemistry and Western blotting. In nontumorigenic WB cells, CD apoptosis was associated with retention of Cx43 in the golgi/ER region of the cytoplasm and decreased GJIC as measured using a preloading fluorescent dye transfer method (calcein AM/DiIC(18)). Cells maintained in CD in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate exhibited restoration of Cx43 at the plasma membrane and increased GJIC and inhibition of apoptosis. These studies show that CD apoptosis in nontumorigenic liver epithelial cells is associated with alterations to Cx43 and GJIC and that an uncoupling of Cx43 localization and GJIC is related to resistance to CD apoptosis in transformed liver epithelial cells. Copyright 2001 Academic Press.

  3. Phenotypic variability in gap junction syndromic skin disorders: experience from KID and Clouston syndromes' clinical diagnostics.

    PubMed

    Kutkowska-Kaźmierczak, Anna; Niepokój, Katarzyna; Wertheim-Tysarowska, Katarzyna; Giza, Aleksandra; Mordasewicz-Goliszewska, Maria; Bal, Jerzy; Obersztyn, Ewa

    2015-08-01

    Connexins belong to the family of gap junction proteins which enable direct cell-to-cell communication by forming channels in adjacent cells. Mutations in connexin genes cause a variety of human diseases and, in a few cases, result in skin disorders. There are significant differences in the clinical picture of two rare autosomal dominant syndromes: keratitis-ichthyosis-deafness (KID) syndrome and hidrotic ectodermal dysplasia (Clouston syndrome), which are caused by GJB2 and GJB6 mutations, respectively. This is despite the fact that, in both cases, malfunctioning of the same family proteins and some overlapping clinical features (nail dystrophy, hair loss, and palmoplantar keratoderma) is observed. KID syndrome is characterized by progressive vascularizing keratitis, ichthyosiform erythrokeratoderma, and neurosensory hearing loss, whereas Clouston syndrome is characterized by nail dystrophy, hypotrichosis, and palmoplantar keratoderma. The present paper presents a Polish patient with sporadic KID syndrome caused by the mutation of p.Asp50Asn in GJB2. The patient encountered difficulties in obtaining a correct diagnosis. The other case presented is that of a family with Clouston syndrome (caused by p.Gly11Arg mutation in GJB6), who are the first reported patients of Polish origin suffering from this disorder. Phenotype diversity among patients with the same genotypes reported to date is also summarized. The conclusion is that proper diagnosis of these syndromes is still challenging and should always be followed by molecular verification.

  4. Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries.

    PubMed

    Kansui, Yasuo; Garland, Christopher J; Dora, Kim A

    2008-08-01

    Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.

  5. Effects of 18beta-glycyrrhetinic acid on the junctional complex and steroidogenesis in rat adrenocortical cells.

    PubMed

    Huang, Shih-Horng; Wu, Jiahn-Chun; Hwang, Ra-Der; Yeo, Hui-Lin; Wang, Seu-Mei

    2003-09-01

    Cellular junctions play important roles in cell differentiation, signal transduction, and cell function. This study investigated their function in steroid secretion by adrenal cells. Immunofluorescence staining revealed the presence of gap junctions and adherens junctions between adrenal cells. The major gap junction protein, connexin43, was seen as a linear dotted pattern of the typical gap junction plaques, in contrast to alpha-, beta-, and gamma-catenin, which were seen as continuous, linear staining of cell-cell adherens junction. Treatment with 18beta-glycyrrhetinic acid, a gap junction inhibitor, reduced the immunoreactivity of these proteins in a time- and dose-dependent manner, and caused the gap junction and adherens junction to separate longitudinally from the cell-cell contact sites, indicating the structural interdependency of these two junctions. Interestingly, 18beta-glycyrrhetinic acid stimulated a two- to three-fold increase in steroid production in these adrenal cells lacking intact cell junctions. These data raise the question of the necessity for cell communication for the endocrine function of adrenal cells. Pharmacological analyses indicated that the steroidogenic effect of 18beta-glycyrrhetinic acid was partially mediated by extracellular signal-related kinase and calcium/calmodulin-dependent kinase, a pathway distinct from the protein kinase A signaling pathway already known to mediate steroidogenesis in adrenal cells. Copyright 2003 Wiley-Liss, Inc.

  6. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    PubMed Central

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-01-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction. PMID:27436542

  7. Gap Junction Regulation of Vascular Tone: Implications of Modulatory Intercellular Communication During Gestation

    PubMed Central

    Ampey, Bryan C.; Morschauser, Timothy J.; Lampe, Paul D.

    2017-01-01

    In the vasculature, gap junctions (GJ) play a multifaceted role by serving as direct conduits for cell–cell intercellular communication via the facilitated diffusion of signaling molecules. GJs are essential for the control of gene expression and coordinated vascular development in addition to vascular function. The coupling of endothelial cells to each other, as well as with vascular smooth muscle cells via GJs, plays a relevant role in the control of vasomotor tone, tissue perfusion and arterial blood pressure. The regulation of cell-signaling is paramount to cardiovascular adaptations of pregnancy. Pregnancy requires highly developed cell-to-cell coupling, which is affected partly through the formation of intercellular GJs by Cx43, a gap junction protein, within adjacent cell membranes to help facilitate the increase of uterine blood flow (UBF) in order to ensure adequate perfusion for nutrient and oxygen delivery to the placenta and thus the fetus. One mode of communication that plays a critical role in regulating Cx43 is the release of endothelial-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) and their respective signaling mechanisms involving second messengers (cAMP and cGMP, respectively) that are likely to be important in maintaining UBF. Therefore, the assertion we present in this review is that GJs play an integral if not a central role in maintaining UBF by controlling rises in vasodilators (PGI2 and NO) via cyclic nucleotides. In this review, we discuss: (1) GJ structure and regulation; (2) second messenger regulation of GJ phosphorylation and formation; (3) pregnancy-induced changes in cell-signaling; and (4) the role of uterine arterial endothelial GJs during gestation. These topics integrate the current knowledge of this scientific field with interpretations and hypotheses regarding the vascular effects that are mediated by GJs and their relationship with vasodilatory vascular adaptations required for modulating the

  8. Molecular Diffusion through Cyanobacterial Septal Junctions.

    PubMed

    Nieves-Morión, Mercedes; Mullineaux, Conrad W; Flores, Enrique

    2017-01-03

    Heterocyst-forming cyanobacteria grow as filaments in which intercellular molecular exchange takes place. During the differentiation of N 2 -fixing heterocysts, regulators are transferred between cells. In the diazotrophic filament, vegetative cells that fix CO 2 through oxygenic photosynthesis provide the heterocysts with reduced carbon and heterocysts provide the vegetative cells with fixed nitrogen. Intercellular molecular transfer has been traced with fluorescent markers, including calcein, 5-carboxyfluorescein, and the sucrose analogue esculin, which are observed to move down their concentration gradient. In this work, we used fluorescence recovery after photobleaching (FRAP) assays in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 to measure the temperature dependence of intercellular transfer of fluorescent markers. We find that the transfer rate constants are directly proportional to the absolute temperature. This indicates that the "septal junctions" (formerly known as "microplasmodesmata") linking the cells in the filament allow molecular exchange by simple diffusion, without any activated intermediate state. This constitutes a novel mechanism for molecular transfer across the bacterial cytoplasmic membrane, in addition to previously characterized mechanisms for active transport and facilitated diffusion. Cyanobacterial septal junctions are functionally analogous to the gap junctions of metazoans. Although bacteria are frequently considered just as unicellular organisms, there are bacteria that behave as true multicellular organisms. The heterocyst-forming cyanobacteria grow as filaments in which cells communicate. Intercellular molecular exchange is thought to be mediated by septal junctions. Here, we show that intercellular transfer of fluorescent markers in the cyanobacterial filament has the physical properties of simple diffusion. Thus, cyanobacterial septal junctions are functionally analogous to metazoan gap junctions

  9. Contractility in type III cochlear fibrocytes is dependent on non-muscle myosin II and intercellular gap junctional coupling.

    PubMed

    Kelly, John J; Forge, Andrew; Jagger, Daniel J

    2012-08-01

    The cochlear spiral ligament is a connective tissue that plays diverse roles in normal hearing. Spiral ligament fibrocytes are classified into functional sub-types that are proposed to carry out specialized roles in fluid homeostasis, the mediation of inflammatory responses to trauma, and the fine tuning of cochlear mechanics. We derived a secondary sub-culture from guinea pig spiral ligament, in which the cells expressed protein markers of type III or "tension" fibrocytes, including non-muscle myosin II (nmII), α-smooth muscle actin (αsma), vimentin, connexin43 (cx43), and aquaporin-1. The cells formed extensive stress fibers containing αsma, which were also associated intimately with nmII expression, and the cells displayed the mechanically contractile phenotype predicted by earlier modeling studies. cx43 immunofluorescence was evident within intercellular plaques, and the cells were coupled via dye-permeable gap junctions. Coupling was blocked by meclofenamic acid (MFA), an inhibitor of cx43-containing channels. The contraction of collagen lattice gels mediated by the cells could be prevented reversibly by blebbistatin, an inhibitor of nmII function. MFA also reduced the gel contraction, suggesting that intercellular coupling modulates contractility. The results demonstrate that these cells can impart nmII-dependent contractile force on a collagenous substrate, and support the hypothesis that type III fibrocytes regulate tension in the spiral ligament-basilar membrane complex, thereby determining auditory sensitivity.

  10. Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition.

    PubMed

    Yang, Yan; Qin, Shu-Kui; Wu, Qiong; Wang, Zi-Shu; Zheng, Rong-Sheng; Tong, Xu-Hui; Liu, Hao; Tao, Liang; He, Xian-Di

    2014-02-01

    Increasing gap junction activity in tumor cells provides a target by which to enhance antineoplastic therapies. Previously, several naturally occurring agents, including all-trans retinoic acid (ATRA) have been demonstrated to increase gap junctional intercellular communication (GJIC) in a number of types of cancer cells. In the present study, we investigated in vitro whether ATRA modulates the response of human hepatocellular carcinoma (HCC) cells to sorafenib, the only proven oral drug for advanced HCC, and the underlying mechanisms. HepG2 and SMMC-7721 cells were treated with sorafenib and/or ATRA, and cell proliferation and apoptosis were analyzed; the role of GJIC was also explored. We found that ATRA, at non-toxic concentrations, enhanced sorafenib-induced growth inhibition in both HCC cell lines, and this effect was abolished by two GJIC inhibitors, 18-α-GA and oleamide. Whereas lower concentrations of sorafenib (5 µM) or ATRA (0.1 or 10 µM) alone modestly induced GJIC activity, the combination of sorafenib plus ATRA resulted in a strong enhancement of GJIC. However, the action paradigm differed in the HepG2 and SMMC-7721 cells, with the dominant effect of GJIC dependent on the cell-specific connexin increase in protein amounts and relocalization. RT-PCR assay further revealed a transcriptional modification of the key structural connexin in the two cell lines. Thus, a connexin-dependent gap junction enhancement may play a central role in ATRA plus sorafenib synergy in inhibiting HCC cell growth. Since both agents are available for human use, the combination treatment represents a future profitable strategy for the treatment of advanced HCC.

  11. Intrathecal injection of carbenoxolone, a gap junction decoupler, attenuates the induction of below-level neuropathic pain after spinal cord injury in rats.

    PubMed

    Roh, Dae-Hyun; Yoon, Seo-Yeon; Seo, Hyoung-Sig; Kang, Suk-Yun; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2010-07-01

    The most common type of chronic pain following spinal cord injury (SCI) is central neuropathic pain and SCI patients typically experience mechanical allodynia and thermal hyperalgesia. The present study was designed to examine the potential role of astrocyte gap junction connectivity in the induction and maintenance of "below-level" neuropathic pain in SCI rats. We examined the effect of intrathecal treatment with carbenoxolone (CARB), a gap junction decoupler, on SCI-induced bilateral thermal hyperalgesia and mechanical allodynia during the induction phase (postoperative days 0 to 5) and the maintenance phase (days 15 to 20) following T13 spinal cord hemisection. Immunohistochemistry was performed to determine potential SCI-induced changes in spinal astrocyte activation and phosphorylation of the NMDA receptor NR1 subunit (pNR1). CARB administered during the induction period dose-dependently attenuated the development of bilateral thermal hyperalgesia and mechanical allodynia. Intrathecal CARB also significantly reduced the bilateral SCI-induced increase in GFAP-immunoreactive (ir) staining and the number of pNR1-ir cell profiles in the spinal cord dorsal horn compared to vehicle-treated rats. In contrast, CARB treatment during the maintenance phase had no effect on the established thermal hyperalgesia and mechanical allodynia nor on spinal GFAP expression or the number of pNR1-ir cell profiles. These results indicate that gap junctions play a critical role in the activation of astrocytes distant from the site of SCI and in the subsequent phosphorylation of NMDA receptors in the lumbar spinal cord. Both of these processes appear to contribute to the induction of bilateral below-level pain in SCI rats. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

    PubMed

    Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

    2015-06-01

    We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

  13. The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

    PubMed Central

    Imbeault, Sophie; Gauvin, Lianne G; Toeg, Hadi D; Pettit, Alexandra; Sorbara, Catherine D; Migahed, Lamiaa; DesRoches, Rebecca; Menzies, A Sheila; Nishii, Kiyomasa; Paul, David L; Simon, Alexander M; Bennett, Steffany AL

    2009-01-01

    Background Gap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells. Results We found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication. Conclusion Together, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells. PMID:19236721

  14. Black Phosphorus-Zinc Oxide Nanomaterial Heterojunction for p-n Diode and Junction Field-Effect Transistor.

    PubMed

    Jeon, Pyo Jin; Lee, Young Tack; Lim, June Yeong; Kim, Jin Sung; Hwang, Do Kyung; Im, Seongil

    2016-02-10

    Black phosphorus (BP) nanosheet is two-dimensional (2D) semiconductor with distinct band gap and attracting recent attention from researches because it has some similarity to gapless 2D semiconductor graphene in the following two aspects: single element (P) for its composition and quite high mobilities depending on its fabrication conditions. Apart from several electronic applications reported with BP nanosheet, here we report for the first time BP nanosheet-ZnO nanowire 2D-1D heterojunction applications for p-n diodes and BP-gated junction field effect transistors (JFETs) with n-ZnO channel on glass. For these nanodevices, we take advantages of the mechanical flexibility of p-type conducting of BP and van der Waals junction interface between BP and ZnO. As a result, our BP-ZnO nanodimension p-n diode displays a high ON/OFF ratio of ∼10(4) in static rectification and shows kilohertz dynamic rectification as well while ZnO nanowire channel JFET operations are nicely demonstrated by BP gate switching in both electrostatics and kilohertz dynamics.

  15. Gap Junction-Mediated Signaling from Motor Neurons Regulates Motor Generation in the Central Circuits of Larval Drosophila.

    PubMed

    Matsunaga, Teruyuki; Kohsaka, Hiroshi; Nose, Akinao

    2017-02-22

    In this study, we used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost in shaking-B 2 ( shakB 2 ) or ogre 2 , gap-junction mutations in Drosophila , or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggested shakB and ogre function in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system. SIGNIFICANCE STATEMENT Motor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the

  16. [The role of gap junction communication in metastatic B16 melanoma in C57BL mice].

    PubMed

    Fëdorov, E S; Manikhas, G M; Petrishchëv, N N; Dubina, M V

    2006-01-01

    The study is concerned with the effects of non-specific blocking gap junction communication with oleamide as well as genesis and spreading of melanoma B16 metastases to the lung in mice C57B1. The blocking exerted no distinct influence on primary tumorigenesis but had a marked effect on metastatic spread. Oleamide treatment during tumor growth led to an increase in area covered by metastases. A correlation was established between metastatic frequency and dosage: 1 mg/kg was followed by an upsurge in frequency of secondary lung tumors while 10 mg/kg--by a drop.

  17. 1.00 MeV proton radiation resistance studies of single-junction and single gap dual-junction amorphous-silicon alloy solar cells

    NASA Technical Reports Server (NTRS)

    Abdulaziz, Salman; Payson, J. S.; Li, Yang; Woodyard, James R.

    1990-01-01

    A comparative study of the radiation resistance of a-Si:H and a-SiGe:H single-junction and a-Si:H dual-junction solar cells was conducted. The cells were irradiated with 1.00-MeV protons with fluences of 1.0 x 10 to the 14th, 5.0 x 10 to the 14th and 1.0 x 10 to the 15th/sq cm and characterized using I-V and quantum efficiency measurements. The radiation resistance of single-junction cells cannot be used to explain the behavior of dual-junction cells at a fluence of 1.0 x 10 to the 15th/sq cm. The a-Si H single-junction cells degraded the least of the three cells; a-SiGe:H single-junction cells showed the largest reduction in short-circuit current, while a-Si:H dual-junction cells exhibited the largest degradation in the open-circuit voltage. The quantum efficiency of the cells degraded more in the red part of the spectrum; the bottom junction degrades first in dual-junction cells.

  18. Burst firing versus synchrony in a gap junction connected olfactory bulb mitral cell network model

    PubMed Central

    O'Connor, Simon; Angelo, Kamilla; Jacob, Tim J. C.

    2012-01-01

    A key player in olfactory processing is the olfactory bulb (OB) mitral cell (MC). We have used dual whole-cell patch-clamp recordings from the apical dendrite and cell soma of MCs to develop a passive compartmental model based on detailed morphological reconstructions of the same cells. Matching the model to traces recorded in experiments we find: Cm = 1.91 ± 0.20 μF cm−2, Rm = 3547 ± 1934 Ω cm2 and Ri = 173 ± 99 Ω cm. We have constructed a six MC gap-junction (GJ) network model of morphologically accurate MCs. These passive parameters (PPs) were then incorporated into the model with Na+, Kdr, and KA conductances and GJs from Migliore et al. (2005). The GJs were placed in the apical dendrite tuft (ADT) and their conductance adjusted to give a coupling ratio between MCs consistent with experimental findings (~0.04). Firing at ~50 Hz was induced in all six MCs with continuous current injections (0.05–0.07 nA) at 20 locations to the ADT of two of the MCs. It was found that MCs in the network synchronized better when they shared identical PPs rather than using their own PPs for the fit suggesting that the OB may have populations of MCs tuned for synchrony. The addition of calcium-activated potassium channels (iKCa) and L-type calcium channels (iCa(L)) (Bhalla and Bower, 1993) to the model enabled MCs to generate burst firing. However, the GJ coupling was no longer sufficient to synchronize firing. When cells were stimulated by a continuous current injection there was an initial period of asynchronous burst firing followed after ~120 ms by synchronous repetitive firing. This occurred as intracellular calcium fell due to reduced iCa(L) activity. The kinetics of one of the iCa(L) gate variables, which had a long activation time constant (τ ~ range 18–150 ms), was responsible for this fall in iCa(L). The model makes predictions about the nature of the kinetics of the calcium current that will need experimental verification. PMID:23060786

  19. Metallic Electrode: Semiconducting Nanotube Junction Model

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Biegel, Bryon (Technical Monitor)

    2001-01-01

    A model is proposed for two observed current-voltage (I-V) patterns in an experiment with a scanning tunneling microscope tip and a carbon nanotube [Collins et al., Science 278, 100 ('97)]. We claim that there are two contact modes for a tip (metal) -nanotube semi conductor) junction depending whether the alignment of the metal and semiconductor band structure is (1) variable (vacuum-gap) or (2) fixed (touching) with V. With the tip grounded, the tunneling case in (1) would produce large dI/dV with V > 0, small dI/dV with V < 0, and I = 0 near V = 0 for an either n- or p-nanotube. However, the Schottky mechanism in (2) would result in forward current with V < 0 for an n-nanotube, while with V > 0 for an p-nanotube. The two observed I-V patterns are thus entirely explained by a tip-nanotube contact of the two types, where the nanotube must be n-type. We apply this picture to the source-drain I-V characteristics in a long nanotube-channel field-effect-transistor (Zhou et al., Appl. Phys. Lett. 76, 1597 ('00)], and show that two independent metal-semiconductor junctions connected in series are responsible for the observed behavior.

  20. mTOR Regulates Gap Junction Alpha-1 Protein Trafficking in Sertoli Cells and Is Required for the Maintenance of Spermatogenesis in Mice.

    PubMed

    Boyer, Alexandre; Girard, Meggie; Thimmanahalli, Dayananda S; Levasseur, Adrien; Céleste, Christophe; Paquet, Marilène; Duggavathi, Rajesha; Boerboom, Derek

    2016-07-01

    The mammalian target of rapamycin (Mtor) gene encodes a serine/threonine kinase that acts as a master regulator of processes as diverse as cell growth, protein synthesis, cytoskeleton reorganization, and cell survival. In the testis, physiological roles for Mtor have been proposed in perinatal Sertoli cell proliferation and blood-testis barrier (BTB) remodeling during spermatogenesis, but no in vivo studies of Mtor function have been reported. Here, we used a conditional knockout approach to target Mtor in Sertoli cells. The resulting Mtor(flox/flox); Amhr2(cre/+) mice were characterized by progressive, adult-onset testicular atrophy associated with disorganization of the seminiferous epithelium, loss of Sertoli cell polarity, increased germ cell apoptosis, premature release of germ cells, decreased epididymal sperm counts, increased sperm abnormalities, and infertility. Histopathologic analysis and quantification of the expression of stage-specific markers showed a specific loss of pachytene spermatocytes and spermatids. Although the BTB and the ectoplasmic specializations did not appear to be altered in Mtor(flox/flox);Amhr2(cre/+) mice, a dramatic redistribution of gap junction alpha-1 (GJA1) was detected in their Sertoli cells. Phosphorylation of GJA1 at Ser373, which is associated with its internalization, was increased in the testes of Mtor(flox/flox); Amhr2(cre/+) mice, as was the expression and phosphorylation of AKT, which phosphorylates GJA1 at this site. Together, these results indicate that Mtor expression in Sertoli cells is required for the maintenance of spermatogenesis and the progression of germ cell development through the pachytene spermatocyte stage. One mechanism of mTOR action may be to regulate gap junction dynamics by inhibiting AKT, thereby decreasing GJA1 phosphorylation and internalization. mTOR regulates gap junction alpha-1 protein distribution in Sertoli cells and is necessary for progression through the pachytene spermatocyte stage.

  1. Precision gap particle separator

    DOEpatents

    Benett, William J.; Miles, Robin; Jones, II., Leslie M.; Stockton, Cheryl

    2004-06-08

    A system for separating particles entrained in a fluid includes a base with a first channel and a second channel. A precision gap connects the first channel and the second channel. The precision gap is of a size that allows small particles to pass from the first channel into the second channel and prevents large particles from the first channel into the second channel. A cover is positioned over the base unit, the first channel, the precision gap, and the second channel. An port directs the fluid containing the entrained particles into the first channel. An output port directs the large particles out of the first channel. A port connected to the second channel directs the small particles out of the second channel.

  2. Tuning of optical and electrical properties of wide band gap Fe:SnO2/Li:NiO p- n junctions using 80 MeV oxygen ion beam

    NASA Astrophysics Data System (ADS)

    Mistry, Bhaumik V.; Avasthi, D. K.; Joshi, U. S.

    2016-12-01

    Electrical and optical properties of pristine and swift heavy ion (SHI) irradiated p- n junction diode have been investigated for advanced electronics application. Fe:SnO2/Li:NiO p- n junction was fabricated by using pulsed laser deposition on c-sapphire substrate. The optical band gaps of Fe:SnO2 and Li:NiO films were obtained to be 3.88 and 3.37 eV, respectively. The current-voltage characteristics of the oxide-based p- n junction showed a rectifying behaviour with turn-on voltage of 0.95 V. The oxide-based p- n junction diode was irradiated to 80 MeV O+6 ions with 1 × 1012 ions/cm2 fluence. Decrease in grain size due to SHI irradiation is confirmed by the grazing angle X-ray diffraction and atomic force microscopy. In comparison with the pristine p- n junction diode, O+6 ion irradiated p-n junction diode shows the increase of surface roughness and decrease of percentage transmittance in visible region. For irradiated p- n junction diode, current-voltage curve has still rectifying behaviour but exhibits lower turn-on voltage than that of virgin p- n junction diode.

  3. Effects of metoprolol therapy on cardiac gap junction remodelling and conduction in human chronic atrial fibrillation

    PubMed Central

    Dhein, S; Rothe, S; Busch, A; Rojas Gomez, DM; Boldt, A; Reutemann, A; Seidel, T; Salameh, A; Pfannmüller, B; Rastan, A; Kostelka, M; Mohr, FW

    2011-01-01

    BACKGROUND AND PURPOSE We investigated the influence of metoprolol on gap junction proteins connexin43 (Cx43) and connexin40 (Cx40) in atrial tissue from patients with/without atrial fibrillation (AF). EXPERIMENTAL APPROACH Left atrial tissue samples from 160 patients with AF or sinus rhythm (SR) with or without metoprolol (mean daily dose: 65.2 ± 9.1 mg·day−1) were analysed for Cx43 and Cx40 by Western blot and immunohistology. Transverse and longitudinal conduction velocities were determined by 64 multi-electrode mapping. KEY RESULTS Both Cx43 and Cx40 expression were significantly increased in patients with AF versus SR. Cx43-expression in AF was significantly higher in patients receiving metoprolol, while Cx40 expression was unaffected by metoprolol treatment. In AF, the ratio of lateral/polar expression of Cx43 and Cx40 was enhanced due to increased expression at the sides of the cells (lateral) and a loss at the cell poles. This AF-induced increase in lateral/polar expression of Cx43, but not of Cx40, was significantly antagonized by metoprolol treatment. Functionally, in AF patients, transverse conduction velocity in atrial samples was significantly enhanced and this change was also significantly antagonized by metoprolol. CONCLUSIONS AND IMPLICATIONS AF induced enhanced lateral expression of Cx43 and Cx40 together with enhanced transverse conduction velocity in left atrial tissue. Alterations in localization of Cx43 and conduction changes were both antagonized by metoprolol, showing that pharmacological modulation of gap junction remodelling seems, in principle, possible. This finding may open new approaches to the development of anti-arrythmic drugs. PMID:21542828

  4. The role of a conserved proline residue in mediating conformational changes associated with voltage gating of Cx32 gap junctions.

    PubMed Central

    Ri, Y; Ballesteros, J A; Abrams, C K; Oh, S; Verselis, V K; Weinstein, H; Bargiello, T A

    1999-01-01

    We have explored the role of a proline residue located at position 87 in the second transmembrane segment (TM2) of gap junctions in the mechanism of voltage-dependent gating of connexin32 (Cx32). Substitution of this proline (denoted Cx32P87) with residues G, A, or V affects channel function in a progressive manner consistent with the expectation that a proline kink (PK) motif exists in the second transmembrane segment (TM2) of this connexin. Mutations of the preceding threonine residue T86 to S, A, C, V, N, or L shift the conductance-voltage relation of wild-type Cx32, such that the mutated channels close at smaller transjunctional voltages. The observed shift in voltage dependence is consistent with a reduction in the open probability of the mutant hemichannels at a transjunctional voltage (Vj) of 0 mV. In both cases in which kinetics were examined, the time constants for reaching steady state were faster for T86N and T86A than for wild type at comparable voltages, suggesting that the T86 mutations cause the energetic destabilization of the open state relative to the other states of the channel protein. The structural underpinnings of the observed effects were explored with Monte Carlo simulations. The conformational space of TM2 helices was found to differ for the T86A, V, N, and L mutants, which produce a less bent helix ( approximately 20 degrees bend angle) compared to the wild type, which has a approximately 37 degrees bend angle. The greater bend angle of the wild-type helix reflects the propensity of the T86 residue to hydrogen bond with the backbone carbonyl of amino acid residue I82. The relative differences in propensity for hydrogen bonding of the mutants relative to the wild-type threonine residue in the constructs we studied (T86A, V, N, L, S, and C) correlate with the shift in the conductance-voltage relation observed for T86 mutations. The data are consistent with a structural model in which the open conformation of the Cx32 channel corresponds to

  5. Communication network in the follicular papilla and connective tissue sheath through gap junctions in human hair follicles.

    PubMed

    Iguchi, Makiko; Hara, Masahiro; Manome, Hideaki; Kobayasi, Hiromi; Tagami, Hachiro; Aiba, Setsuya

    2003-06-01

    Epithelial-mesenchymal interactions play a crucial role in the induction of life-long cyclic transformations of hair follicles. Many studies have already demonstrated several candidates for the soluble factors secreted from the mesenchymal components of the hair follicle, i.e. the follicular papilla (FP) and connective tissue sheath (CTS), which may be responsible for hair cycling. In this paper, we focused on cell-cell contact between FP cells (FPCs), between CTS cells (CTSCs), and between FPCs and CTSCs that may allow these mesenchymal components to function as a syncytium during hair cycling. Electron microscopic examination of the FP and the CTS obtained from human scalp revealed a tri-lamellar structure of the plasma membranes, which is a characteristic of gap junctions at the cell-cell contacting area. The immunohistochemical study with anticonnexin 43 Ab using a confocal laser scanning microscope demonstrated numerous spotted positive signals scattered throughout the FP. In the CTS, spotted positive signals were arranged linearly along the basement membrane of the hair follicle. In particular, these positive spots were aggregated in the transitional region between the FP and the CTS. By Western blot analysis of total protein extracts from the cultured FPCs and neonatal human dermal fibroblasts using anticonnexin 43 antibody, a positive band corresponding to connexin 43 was detected at 43 kDa on both the FPC lane and fibroblast lane. These findings suggest that the FP and the CTS form a communicating network through gap junctions, which may play a role in controlling the dynamic structural changes of hair follicles during hair cycling.

  6. Competitive behavior of photons contributing to junction voltage jump in narrow band-gap semiconductor multi-quantum-well laser diodes at lasing threshold

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feng, Liefeng, E-mail: fengliefeng@tju.edu.cn, E-mail: lihongru@nankai.edu.cn; Yang, Xiufang; Wang, Cunda

    2015-04-15

    The junction behavior of different narrow band-gap multi-quantum-well (MQW) laser diodes (LDs) confirmed that the jump in the junction voltage in the threshold region is a general characteristic of narrow band-gap LDs. The relative change in the 1310 nm LD is the most obvious. To analyze this sudden voltage change, the threshold region is divided into three stages by I{sub th}{sup l} and I{sub th}{sup u}, as shown in Fig. 2; I{sub th}{sup l} is the conventional threshold, and as long as the current is higher than this threshold, lasing exists and the IdV/dI-I plot drops suddenly; I{sub th}{sup u}more » is the steady lasing point, at which the separation of the quasi-Fermi levels of electron and holes across the active region (V{sub j}) is suddenly pinned. Based on the evolutionary model of dissipative structure theory, the rate equations of the photons in a single-mode LD were deduced in detail at I{sub th}{sup l} and I{sub th}{sup u}. The results proved that the observed behavior of stimulated emission suddenly substituting for spontaneous emission, in a manner similar to biological evolution, must lead to a sudden increase in the injection carriers in the threshold region, which then causes the sudden increase in the junction voltage in this region.« less

  7. Comparative effects of torasemide and furosemide on gap junction proteins and cardiac fibrosis in a rat model of dilated cardiomyopathy.

    PubMed

    Watanabe, Kenichi; Sreedhar, Remya; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Giridharan, Vijayasree V; Antony, Shanish; Harima, Meilei; Nakamura, Masahiko; Suzuki, Kenji; Suzuki, Hiroshi; Sone, Hirohito; Arumugam, Somasundaram

    2017-03-01

    Cardiac fibrosis is the major hallmark of adverse cardiac remodeling in chronic heart failure (CHF) and its therapeutic targeting might help against cardiac dysfunction during chronic conditions. Diuretic agents are potentially useful in these cases, but their effects on the cardiac fibrosis pathogenesis are yet to be identified. This study was designed to identify and compare the effects of diuretic drugs torasemide and furosemide on cardiac fibrosis in a rat model of dilated cardiomyopathy induced by porcine cardiac myosin mediated experimental autoimmune myocarditis. Gap junction proteins, connexin-43 and N-cadherin, expressions were downregulated in the hearts of CHF rats, while torasemide treatment has upregulated their expression. Western blotting and immunohistochemical analysis for various cardiac fibrosis related proteins as well as histopathological studies have shown that both drugs have potential anti-fibrotic effects. Among them, torasemide has superior efficacy in offering protection against adverse cardiac remodeling in the selected rat model of dilated cardiomyopathy. In conclusion, torasemide treatment has potential anti-fibrotic effect in the hearts of CHF rats, possibly via improving the gap junction proteins expression and thereby improving the cell-cell interaction in the heart. © 2016 BioFactors, 43(2):187-194, 2017. © 2016 International Union of Biochemistry and Molecular Biology.

  8. Complementary junction heterostructure field-effect transistor

    DOEpatents

    Baca, Albert G.; Drummond, Timothy J.; Robertson, Perry J.; Zipperian, Thomas E.

    1995-01-01

    A complimentary pair of compound semiconductor junction heterostructure field-effect transistors and a method for their manufacture are disclosed. The p-channel junction heterostructure field-effect transistor uses a strained layer to split the degeneracy of the valence band for a greatly improved hole mobility and speed. The n-channel device is formed by a compatible process after removing the strained layer. In this manner, both types of transistors may be independently optimized. Ion implantation is used to form the transistor active and isolation regions for both types of complimentary devices. The invention has uses for the development of low power, high-speed digital integrated circuits.

  9. Complementary junction heterostructure field-effect transistor

    DOEpatents

    Baca, A.G.; Drummond, T.J.; Robertson, P.J.; Zipperian, T.E.

    1995-12-26

    A complimentary pair of compound semiconductor junction heterostructure field-effect transistors and a method for their manufacture are disclosed. The p-channel junction heterostructure field-effect transistor uses a strained layer to split the degeneracy of the valence band for a greatly improved hole mobility and speed. The n-channel device is formed by a compatible process after removing the strained layer. In this manner, both types of transistors may be independently optimized. Ion implantation is used to form the transistor active and isolation regions for both types of complimentary devices. The invention has uses for the development of low power, high-speed digital integrated circuits. 10 figs.

  10. INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION BY PERFLUORINATED COMPOUNDS IN RAT LIVER AND DOLPHIN KIDNEY EPITHELIAL CELL LINES IN VITRO AND SPRAGUE-DAWLEY RATS IN VIVO

    EPA Science Inventory

    Abstract

    Gap Junctional Intercellular Communication (GJIC) is the major pathway of intercellular signal transduction, and is, thus, important for normal cell growth and function. Recent studies have revealed a global distribution of some perfluorinated organic compounds e...

  11. Gap Junctions Are Involved in the Rescue of CFTR-Dependent Chloride Efflux by Amniotic Mesenchymal Stem Cells in Coculture with Cystic Fibrosis CFBE41o- Cells.

    PubMed

    Carbone, Annalucia; Zefferino, Roberto; Beccia, Elisa; Casavola, Valeria; Castellani, Stefano; Di Gioia, Sante; Giannone, Valentina; Seia, Manuela; Angiolillo, Antonella; Colombo, Carla; Favia, Maria; Conese, Massimo

    2018-01-01

    We previously found that human amniotic mesenchymal stem cells (hAMSCs) in coculture with CF immortalised airway epithelial cells (CFBE41o- line, CFBE) on Transwell® filters acquired an epithelial phenotype and led to the expression of a mature and functional CFTR protein. In order to explore the role of gap junction- (GJ-) mediated intercellular communication (GJIC) in this rescue, cocultures (hAMSC : CFBE, 1 : 5 ratio) were studied for the formation of GJIC, before and after silencing connexin 43 (Cx43), a major component of GJs. Functional GJs in cocultures were inhibited when the expression of the Cx43 protein was downregulated. Transfection of cocultures with siRNA against Cx43 resulted in the absence of specific CFTR signal on the apical membrane and reduction in the mature form of CFTR (band C), and in parallel, the CFTR-dependent chloride channel activity was significantly decreased. Cx43 downregulation determined also a decrease in transepithelial resistance and an increase in paracellular permeability as compared with control cocultures, implying that GJIC may regulate CFTR expression and function that in turn modulate airway epithelium tightness. These results indicate that GJIC is involved in the correction of CFTR chloride channel activity upon the acquisition of an epithelial phenotype by hAMSCs in coculture with CF cells.

  12. Low Connexin Channel-Dependent Intercellular Communication in Human Adult Hematopoietic Progenitor/Stem Cells: Probing Mechanisms of Autologous Stem Cell Therapy

    PubMed Central

    Yang, Jian; Darley, Richard L; Hallett, Maurice; Evans, W Howard

    2009-01-01

    Human bone marrow is a clinical source of autologous progenitor stem cells showing promise for cardiac repair following ischemic insult. Functional improvements following delivery of adult bone marrow CD34+ cells into heart tissue may require metabolic/electrical communication between participating cells. Since connexin43 (Cx43) channels are implicated in cardiogenesis and provide intercellular connectivity in the heart, the authors analyzed the expression of 20 connexins (Cx) in CD34+ cells and in monocytes and granulocytes in bone marrow and spinal cord. Reverse transcriptase-polymerase chain reaction (RT-PCR) detected only low expression of Cx43 and Cx37. Very low level dye coupling was detected by flow cytometry between CD34+ cells and other Cx43 expressing cells, including HL-1 cardiac cells, and was not inhibited by specific gap junction inhibitors. The results indicate that CD34+ cells are unlikely to communicate via gap junctions and the authors conclude that use of CD34+ cells to repair damaged hearts is unlikely to involve gap junctions. The results concur with the hypothesis that bone marrow cells elicit improved cardiac function through release of undefined paracrine mediators. PMID:20298144

  13. Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

    PubMed Central

    Zhang, Yiyao; Isayev, Orkhan; Heilmann, Katharina; Schoensiegel, Frank; Liu, Li; Nessling, Michelle; Richter, Karsten; Labsch, Sabrina; Nwaeburu, Clifford C.; Mattern, Juergen; Gladkich, Jury; Giese, Nathalia; Werner, Jens; Schemmer, Peter; Gross, Wolfgang; Gebhard, Martha M.; Gerhauser, Clarissa; Schaefer, Michael; Herr, Ingrid

    2014-01-01

    The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA. PMID:24742583

  14. Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication.

    PubMed

    Forster, Tobias; Rausch, Vanessa; Zhang, Yiyao; Isayev, Orkhan; Heilmann, Katharina; Schoensiegel, Frank; Liu, Li; Nessling, Michelle; Richter, Karsten; Labsch, Sabrina; Nwaeburu, Clifford C; Mattern, Juergen; Gladkich, Jury; Giese, Nathalia; Werner, Jens; Schemmer, Peter; Gross, Wolfgang; Gebhard, Martha M; Gerhauser, Clarissa; Schaefer, Michael; Herr, Ingrid

    2014-03-30

    The extreme aggressiveness of pancreatic ductal adenocarcinoma (PDA) has been associated with blocked gap junctional intercellular communication (GJIC) and the presence of cancer stem cells (CSCs). We examined whether disturbed GJIC is responsible for a CSC phenotype in established and primary cancer cells and patient tissue of PDA using interdisciplinary methods based in physiology, cell and molecular biology, histology and epigenetics. Flux of fluorescent dyes and gemcitabine through gap junctions (GJs) was intact in less aggressive cells but not in highly malignant cells with morphological dysfunctional GJs. Among several connexins, only Cx43 was expressed on the cell surface of less aggressive and GJIC-competent cells, whereas Cx43 surface expression was absent in highly malignant, E-cadherin-negative and GJIC-incompetent cells. The levels of total Cx43 protein and Cx43 phosphorylated at Ser368 and Ser279/282 were high in normal tissue but low to absent in malignant tissue. si-RNA-mediated inhibition of Cx43 expression in GJIC-competent cells prevented GJIC and induced colony formation and the expression of stem cell-related factors. The bioactive substance sulforaphane enhanced Cx43 and E-cadherin levels, inhibited the CSC markers c-Met and CD133, improved the functional morphology of GJs and enhanced GJIC. Sulforaphane altered the phosphorylation of several kinases and their substrates and inhibition of GSK3, JNK and PKC prevented sulforaphane-induced CX43 expression. The sulforaphane-mediated expression of Cx43 was not correlated with enhanced Cx43 RNA expression, acetylated histone binding and Cx43 promoter de-methylation, suggesting that posttranslational phosphorylation is the dominant regulatory mechanism. Together, the absence of Cx43 prevents GJIC and enhances aggressiveness, whereas sulforaphane counteracts this process, and our findings highlight dietary co-treatment as a viable treatment option for PDA.

  15. Shalbatana/Simud Vallis Junction

    NASA Image and Video Library

    2003-01-11

    The sinuous channels and streamlined islands at the junction of Shalbatana and Simud Vallis, seen in this NASA Mars Odyssey image, present an erosional history of the catastrophic floods that scoured the Martian surface hundreds of millions of years ago.

  16. Redox-mediated regulation of connexin proteins; focus on nitric oxide.

    PubMed

    García, Isaac E; Sánchez, Helmuth A; Martínez, Agustín D; Retamal, Mauricio A

    2018-01-01

    Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

    PubMed

    Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

    2016-09-01

    Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Modulation of connexin expression and gap junction communication in astrocytes by the gram-positive bacterium S. aureus.

    PubMed

    Esen, Nilufer; Shuffield, Debbie; Syed, Mohsin M D; Kielian, Tammy

    2007-01-01

    Gap junctions establish direct intercellular conduits between adjacent cells and are formed by the hexameric organization of protein subunits called connexins (Cx). It is unknown whether the proinflammatory milieu that ensues during CNS infection with S. aureus, one of the main etiologic agents of brain abscess in humans, is capable of eliciting regional changes in astrocyte homocellular gap junction communication (GJC) and, by extension, influencing neuron homeostasis at sites distant from the primary focus of infection. Here we investigated the effects of S. aureus and its cell wall product peptidoglycan (PGN) on Cx43, Cx30, and Cx26 expression, the main Cx isoforms found in astrocytes. Both bacterial stimuli led to a time-dependent decrease in Cx43 and Cx30 expression; however, Cx26 levels were elevated following bacterial exposure. Functional examination of dye coupling, as revealed by single-cell microinjections of Lucifer yellow, demonstrated that both S. aureus and PGN inhibited astrocyte GJC. Inhibition of protein synthesis with cyclohexamide (CHX) revealed that S. aureus directly modulates, in part, Cx43 and Cx30 expression, whereas Cx26 levels appear to be regulated by a factor(s) that requires de novo protein production; however, CHX did not alter the inhibitory effects of S. aureus on astrocyte GJC. The p38 MAPK inhibitor SB202190 was capable of partially restoring the S. aureus-mediated decrease in astrocyte GJC to that of unstimulated cells, suggesting the involvement of p38 MAPK-dependent pathway(s). These findings could have important implications for limiting the long-term detrimental effects of abscess formation in the brain which may include seizures and cognitive deficits. Copyright 2006 Wiley-Liss, Inc.

  19. Closing the gap between glia and neuroblast proliferation.

    PubMed

    Limmer, Stefanie; Klämbt, Christian

    2014-08-11

    Reporting in this issue of Developmental Cell, Spéder and Brand (2014) show that gap junctions are required in blood-brain barrier glial cells to reactivate proliferation of quiescent neuroblasts. Gap junctions allow synchronous Ca(2+) waves and control insulin-like protein Dipl6 expression and secretion to trigger neuroblast division. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Influence of the spatially inhomogeneous gap distribution on the quasiparticle current in c-axis junctions involving d-wave superconductors with charge density waves.

    PubMed

    Ekino, T; Gabovich, A M; Suan Li, Mai; Szymczak, H; Voitenko, A I

    2016-11-09

    The quasiparticle tunnel current J(V) between the superconducting ab-planes along the c-axis and the corresponding conductance [Formula: see text] were calculated for symmetric junctions composed of disordered d-wave layered superconductors partially gapped by charge density waves (CDWs). Here, V is the voltage. Both the checkerboard and unidirectional CDWs were considered. It was shown that the spatial spread of the CDW-pairing strength substantially smears the peculiarities of G(V) appropriate to uniform superconductors. The resulting curves G(V) become very similar to those observed for a number of cuprates in intrinsic junctions, e.g. mesas. In particular, the influence of CDWs may explain the peak-dip-hump structures frequently found for high-T c oxides.

  1. Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication

    PubMed Central

    Rovetta, Francesca; Boniotti, Jennifer; Mazzoleni, Giovanna

    2017-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction. PMID:28607928

  2. ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

    PubMed Central

    Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

    2017-01-01

    ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

  3. Field-free junctions for surface electrode ion traps

    NASA Astrophysics Data System (ADS)

    Jordens, Robert; Schmied, R.; Blain, M. G.; Leibfried, D.; Wineland, D.

    2015-05-01

    Intersections between transport guides in a network of RF ion traps are a key ingredient to many implementations of scalable quantum information processing with trapped ions. Several junction architectures demonstrated so far are limited by varying radial secular frequencies, a reduced trap depth, or a non-vanishing RF field along the transport channel. We report on the design and progress in implementing a configurable microfabricated surface electrode Y-junction that employs switchable RF electrodes. An essentially RF-field-free pseudopotential guide between any two legs of the junction can be established by applying RF potential to a suitable pair of electrodes. The transport channel's height above the electrodes, its depth and radial curvature are constant to within 15%. Supported by IARPA, Sandia, NSA, ONR, and the NIST Quantum Information Program.

  4. Effect of reabsorbed recombination radiation on the saturation current of direct gap p-n junctions

    NASA Technical Reports Server (NTRS)

    Von Roos, O.; Mavromatis, H.

    1984-01-01

    The application of the radiative transfer theory for semiconductors to p-n homojunctions subject to low level injection conditions is discussed. By virtue of the interaction of the radiation field with free carriers across the depletion layer, the saturation current density in Shockley's expression for the diode current is reduced at high doping levels. The reduction, due to self-induced photon generation, is noticeable for n-type material owing to the small electron effective mass in direct band-gap III-V compounds. The effect is insignificant in p-type material. At an equilibrium electron concentration of 2 x 10 to the 18th/cu cm in GaAs, a reduction of the saturation current density by 15 percent is predicted. It is concluded that realistic GaAs p-n junctions possess a finite thickness.

  5. Effects of fluctuations on electrical properties of gap-junction connected cells in the turtle retina.

    PubMed

    Louis, E; Degli Esposti Boschi, C; Ortega, G; Andreu, E; Fernández, E; Sánchez-Andrés, J V

    2002-04-19

    Electrical properties of gap-junction connected cells (input voltage and length constant) are shown to depend strongly on fluctuations in membrane and contact conductances. This opens new possibilities and incorporates a further difficulty to the analysis of electrophysiological data, since four, instead of two, parameters (the average values and the magnitude of fluctuations of the two conductances) have to be used in fitting the experimental data. The discussion is illustrated by investigating the effects of dopamine on signal spreading in horizontal cells of turtle retina, assuming a linear cell arrangement. It is shown that while a standard fitting with the average values of the two conductances leads to the conclusion that both are equally affected by dopamine, including fluctuations allows fitting the data by varying just the average contact conductance plus the magnitude of fluctuations.

  6. Muscle gap approach under a minimally invasive channel technique for treating long segmental lumbar spinal stenosis

    PubMed Central

    Bin, Yang; De cheng, Wang; wei, Wang Zong; Hui, Li

    2017-01-01

    Abstract This study aimed to compare the efficacy of muscle gap approach under a minimally invasive channel surgical technique with the traditional median approach. In the Orthopedics Department of Traditional Chinese and Western Medicine Hospital, Tongzhou District, Beijing, 68 cases of lumbar spinal canal stenosis underwent surgery using the muscle gap approach under a minimally invasive channel technique and a median approach between September 2013 and February 2016. Both approaches adopted lumbar spinal canal decompression, intervertebral disk removal, cage implantation, and pedicle screw fixation. The operation time, bleeding volume, postoperative drainage volume, and preoperative and postoperative visual analog scale (VAS) score and Japanese Orthopedics Association score (JOA) were compared between the 2 groups. All patients were followed up for more than 1 year. No significant difference between the 2 groups was found with respect to age, gender, surgical segments. No diversity was noted in the operation time, intraoperative bleeding volume, preoperative and 1 month after the operation VAS score, preoperative and 1 month after the operation JOA score, and 6 months after the operation JOA score between 2 groups (P > .05). The amount of postoperative wound drainage (260.90 ± 160 mL vs 447.80 ± 183.60 mL, P < .001) and the VAS score 6 months after the operation (1.71 ± 0.64 vs 2.19 ± 0.87, P = .01) were significantly lower in the muscle gap approach group than in the median approach group (P < .05). In the muscle gap approach under a minimally invasive channel group, the average drainage volume was reduced by 187 mL, and the average VAS score 6 months after the operation was reduced by an average of 0.48. The muscle gap approach under a minimally invasive channel technique is a feasible method to treat long segmental lumbar spinal canal stenosis. It retains the integrity of the posterior spine complex to the greatest

  7. NbN tunnel junctions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Villegier, J.C.; Goniche, M.; Renard, P.

    1985-03-01

    All-niobium nitride Josephson junctions have been prepared successfully using a new processing called SNOP: Selective Niobium (Nitride) Overlap Process. Such a process involves the ''trilayer'' deposition on the whole wafer before selective patterning of the electrodes by optically controlled Dry Reactive Ion Etching. Only two photomask levels are need to define an ''overlap'' or a ''cross-type'' junction with a good accuracy. The properties of the niobium nitride films deposited by DC-Magnetron sputtering and the surface oxide growth are analysed. The most critical point to obtain high quality and high gap value junctions resides in the early stage of the NbNmore » counterelectrode growth. Some possibilities to overcome such a handicap exist even if the fabrication needs substrate temperatures below 250/sup 0/C.« less

  8. Tumor-induced loss of mural Connexin 43 gap junction activity promotes endothelial proliferation.

    PubMed

    Choudhary, Mayur; Naczki, Christine; Chen, Wenhong; Barlow, Keith D; Case, L Douglas; Metheny-Barlow, Linda J

    2015-05-23

    Proper functional association between mural cells and endothelial cells (EC) causes EC of blood vessels to become quiescent. Mural cells on tumor vessels exhibit decreased attachment to EC, which allows vessels to be unstable and proliferative. The mechanisms by which tumors prevent proper association between mural cells and EC are not well understood. Since gap junctions (GJ) play an important role in cell-cell contact and communication, we investigated whether loss of GJ plays a role in tumor-induced mural cell dissociation. Mural cell regulation of endothelial proliferation was assessed by direct co-culture assays of fluorescently labeled cells quantified by flow cytometry or plate reader. Gap junction function was assessed by parachute assay. Connexin 43 (Cx43) protein in mural cells exposed to conditioned media from cancer cells was assessed by Western and confocal microscopy; mRNA levels were assessed by quantitative real-time PCR. Expression vectors or siRNA were utilized to overexpress or knock down Cx43. Tumor growth and angiogenesis was assessed in mouse hosts deficient for Cx43. Using parachute dye transfer assay, we demonstrate that media conditioned by MDA-MB-231 breast cancer cells diminishes GJ communication between mural cells (vascular smooth muscle cells, vSMC) and EC. Both protein and mRNA of the GJ component Connexin 43 (Cx43) are downregulated in mural cells by tumor-conditioned media; media from non-tumorigenic MCF10A cells had no effect. Loss of GJ communication by Cx43 siRNA knockdown, treatment with blocking peptide, or exposure to tumor-conditioned media diminishes the ability of mural cells to inhibit EC proliferation in co-culture assays, while overexpression of Cx43 in vSMC restores GJ and endothelial inhibition. Breast tumor cells implanted into mice heterozygous for Cx43 show no changes in tumor growth, but exhibit significantly increased tumor vascularization determined by CD31 staining, along with decreased mural cell support

  9. Behavior of a chemically doped graphene junction

    NASA Astrophysics Data System (ADS)

    Farmer, Damon B.; Lin, Yu-Ming; Afzali-Ardakani, Ali; Avouris, Phaedon

    2009-05-01

    Polyethylene imine and diazonium salts are used as complementary molecular dopants to engineer a doping profile in a graphene transistor. Electronic transport in this device reveals the presence of two distinct resistance maxima, alluding to neutrality point separation and subsequent formation of a spatially abrupt junction. Carrier mobility in this device is not significantly affected by molecular doping or junction formation, and carrier transmission is found to scale inversely with the effective channel length of the device. Chemical dilutions are used to modify the dopant concentration and, in effect, alter the properties of the junction.

  10. Dispersion mechanisms of a tidal river junction in the Sacramento–San Joaquin Delta, California

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gleichauf, Karla T.; Wolfram, Philip J.; Monsen, Nancy E.

    In branching channel networks, such as in the Sacramento–San Joaquin River Delta, junction flow dynamics contribute to dispersion of ecologically important entities such as fish, pollutants, nutrients, salt, sediment, and phytoplankton. Flow transport through a junction largely arises from velocity phasing in the form of divergent flow between junction channels for a portion of the tidal cycle. Field observations in the Georgiana Slough junction, which is composed of the North and South Mokelumne rivers, Georgiana Slough, and the Mokelumne River, show that flow phasing differences between these rivers arise from operational, riverine, and tidal forcing. A combination of Acoustic Dopplermore » Current Profile (ADCP) boat transecting and moored ADCPs over a spring–neap tidal cycle (May to June 2012) monitored the variability of spatial and temporal velocity, respectively. Two complementary drifter studies enabled assessment of local transport through the junction to identify small-scale intrajunction dynamics. We supplemented field results with numerical simulations using the SUNTANS model to demonstrate the importance of phasing offsets for junction transport and dispersion. Different phasing of inflows to the junction resulted in scalar patchiness that is characteristic of MacVean and Stacey’s (2011) advective tidal trapping. Furthermore, we observed small-scale junction flow features including a recirculation zone and shear layer, which play an important role in intra-junction mixing over time scales shorter than the tidal cycle (i.e., super-tidal time scales). Thus, the study period spanned open- and closed-gate operations at the Delta Cross Channel. Synthesis of field observations and modeling efforts suggest that management operations related to the Delta Cross Channel can strongly affect transport in the Delta by modifying the relative contributions of tidal and riverine flows, thereby changing the junction flow phasing.« less

  11. Dispersion mechanisms of a tidal river junction in the Sacramento–San Joaquin Delta, California

    DOE PAGES

    Gleichauf, Karla T.; Wolfram, Philip J.; Monsen, Nancy E.; ...

    2014-12-17

    In branching channel networks, such as in the Sacramento–San Joaquin River Delta, junction flow dynamics contribute to dispersion of ecologically important entities such as fish, pollutants, nutrients, salt, sediment, and phytoplankton. Flow transport through a junction largely arises from velocity phasing in the form of divergent flow between junction channels for a portion of the tidal cycle. Field observations in the Georgiana Slough junction, which is composed of the North and South Mokelumne rivers, Georgiana Slough, and the Mokelumne River, show that flow phasing differences between these rivers arise from operational, riverine, and tidal forcing. A combination of Acoustic Dopplermore » Current Profile (ADCP) boat transecting and moored ADCPs over a spring–neap tidal cycle (May to June 2012) monitored the variability of spatial and temporal velocity, respectively. Two complementary drifter studies enabled assessment of local transport through the junction to identify small-scale intrajunction dynamics. We supplemented field results with numerical simulations using the SUNTANS model to demonstrate the importance of phasing offsets for junction transport and dispersion. Different phasing of inflows to the junction resulted in scalar patchiness that is characteristic of MacVean and Stacey’s (2011) advective tidal trapping. Furthermore, we observed small-scale junction flow features including a recirculation zone and shear layer, which play an important role in intra-junction mixing over time scales shorter than the tidal cycle (i.e., super-tidal time scales). Thus, the study period spanned open- and closed-gate operations at the Delta Cross Channel. Synthesis of field observations and modeling efforts suggest that management operations related to the Delta Cross Channel can strongly affect transport in the Delta by modifying the relative contributions of tidal and riverine flows, thereby changing the junction flow phasing.« less

  12. 75 FR 76294 - Radio Broadcasting Services: Pacific Junction, IA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-08

    ... FEDERAL COMMUNICATIONS COMMISSION 47 CFR Part 73 [DA 10-2236; MB Docket No. 10-108] Radio Broadcasting Services: Pacific Junction, IA AGENCY: Federal Communications Commission. ACTION: Final rule. SUMMARY: The staff deletes FM Channel 299C2 at Pacific Junction, Iowa, because the record in this...

  13. Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

    PubMed Central

    Higgins, Adam Z.; Karlsson, Jens O.M.

    2013-01-01

    The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell

  14. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

    PubMed

    Kang, Bok Eum; Baker, Bradley J

    2016-04-04

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response.

  15. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions

    PubMed Central

    Kang, Bok Eum; Baker, Bradley J.

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  16. Disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis.

    PubMed

    Markoullis, Kyriaki; Sargiannidou, Irene; Gardner, Christopher; Hadjisavvas, Andreas; Reynolds, Richard; Kleopa, Kleopas A

    2012-07-01

    Gap junctions (GJs) are vital for oligodendrocyte survival and myelination. In order to examine how different stages of inflammatory demyelination affect oligodendrocyte GJs, we studied the expression of oligodendrocytic connexin32 (Cx32) and Cx47 and astrocytic Cx43 in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS) induced by recombinant myelin oligodendrocyte glycoprotein. EAE was characterized by remissions and relapses with demyelination and axonal loss. Formation of GJ plaques was quantified in relation to the lesions and in normal appearing white matter (NAWM). During acute EAE at 14 days postimmunization (dpi) both Cx47 and Cx32 GJs were severely reduced within and around lesions but also in the NAWM. Cx47 was localized intracellularly in oligodendrocytes while protein levels remained unchanged, and this redistribution coincided with the loss of Cx43 GJs in astrocytes. Cx47 and Cx32 expression increased during remyelination at 28 dpi but decreased again at 50 dpi in the relapsing phase. Oligodendrocyte GJs remained reduced even in NAWM, despite increased formation of Cx43 GJs toward lesions indicating astrogliosis. EAE induced in Cx32 knockout mice resulted in an exacerbated clinical course with more demyelination and axonal loss compared with wild-type EAE mice of the same backcross, despite similar degree of inflammation, and an overall milder loss of Cx47 and Cx43 GJs. Thus, EAE causes persistent impairment of both intra- and intercellular oligodendrocyte GJs even in the NAWM, which may be an important mechanism of MS progression. Furthermore, GJ deficient myelinated fibers appear more vulnerable to CNS inflammatory demyelination. Copyright © 2012 Wiley Periodicals, Inc.

  17. Gap junctional intercellular communication dysfunction mediates the cognitive impairment induced by cerebral ischemia-reperfusion injury: PI3K/Akt pathway involved.

    PubMed

    Zhou, Shujun; Fang, Zheng; Wang, Gui; Wu, Song

    2017-01-01

    Cerebral ischemia/reperfusion (I/R) injury causes hippocampal apoptosis and cognitive impairment, and the dysfunction of gap junction intercellular communication (GJIC) may contribute to the cognitive impairment. We aim to examine the impact of cerebral I/R injury on cognitive impairment, the role of GJIC dysfunction in the rat hippocampus and the involvement of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway. Rats were subjected to a cerebral I/R procedure and underwent cognitive assessment with the novel object recognition and Morris Water Maze tasks. The distance of Lucifer Yellow dye transfer and the Cx43 protein were examined to measure GJIC. Neural apoptosis was assessed with the terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. After rats received inhibitors of the PI3K/Akt pathway, GJIC and cognitive ability were measured again. GJIC promotion by ZP123 significantly reversed cognitive impairment and hippocampal apoptosis induced by cerebral I/R, while the inhibition of GJIC by octanol significantly facilitated cognitive impairment and hippocampal apoptosis. The phosphorylation of Akt was enhanced by cerebral I/R and octanol but inhibited by ZP123. The inhibition of the PI3K/Akt pathway significantly suppressed GJIC and cognitive impairment. The PI3K/Akt pathway is involved in cognitive impairment caused by gap junctional communication dysfunction in the rat hippocampus after ischemia-reperfusion injury.

  18. Effects of gap junction inhibition on contraction waves in the murine small intestine in relation to coupled oscillator theory.

    PubMed

    Parsons, Sean P; Huizinga, Jan D

    2015-02-15

    Waves of contraction in the small intestine correlate with slow waves generated by the myenteric network of interstitial cells of Cajal. Coupled oscillator theory has been used to explain steplike gradients in the frequency (frequency plateaux) of contraction waves along the length of the small intestine. Inhibition of gap junction coupling between oscillators should lead to predictable effects on these plateaux and the wave dislocation (wave drop) phenomena associated with their boundaries. It is these predictions that we wished to test. We used a novel multicamera diameter-mapping system to measure contraction along 25- to 30-cm lengths of murine small intestine. There were typically two to three plateaux per length of intestine. Dislocations could be limited to the wavefronts immediately about the terminated wave, giving the appearance of a three-pronged fork, i.e., a fork dislocation; additionally, localized decreases in velocity developed across a number of wavefronts, ending with the terminated wave, which could appear as a fork, i.e., slip dislocations. The gap junction inhibitor carbenoxolone increased the number of plateaux and dislocations and decreased contraction wave velocity. In some cases, the usual frequency gradient was reversed, with a plateau at a higher frequency than its proximal neighbor; thus fork dislocations were inverted, and the direction of propagation was reversed. Heptanol had no effect on the frequency or velocity of contractions but did reduce their amplitude. To understand intestinal motor patterns, the pacemaker network of the interstitial cells of Cajal is best evaluated as a system of coupled oscillators. Copyright © 2015 the American Physiological Society.

  19. Electron optics with ballistic graphene junctions

    NASA Astrophysics Data System (ADS)

    Chen, Shaowen

    Electrons transmitted across a ballistic semiconductor junction undergo refraction, analogous to light rays across an optical boundary. A pn junction theoretically provides the equivalent of a negative index medium, enabling novel electron optics such as negative refraction and perfect (Veselago) lensing. In graphene, the linear dispersion and zero-gap bandstructure admit highly transparent pn junctions by simple electrostatic gating, which cannot be achieved in conventional semiconductors. Robust demonstration of these effects, however, has not been forthcoming. Here we employ transverse magnetic focusing to probe propagation across an electrostatically defined graphene junction. We find perfect agreement with the predicted Snell's law for electrons, including observation of both positive and negative refraction. Resonant transmission across the pn junction provides a direct measurement of the angle dependent transmission coefficient, and we demonstrate good agreement with theory. Comparing experimental data with simulation reveals the crucial role played by the effective junction width, providing guidance for future device design. Efforts toward sharper pn junction and possibility of zero field Veselago lensing will also be discussed. This work is supported by the Semiconductor Research Corporations NRI Center for Institute for Nanoelectronics Discovery and Exploration (INDEX).

  20. Fabrication and characterization of high current-density, submicron, NbN/MgO/NbN tunnel junctions

    NASA Technical Reports Server (NTRS)

    Stern, J. A.; Leduc, Henry G.; Judas, A. J.

    1992-01-01

    At near-millimeter wavelengths, heterodyne receivers based on SIS tunnel junctions are the most sensitive available. However, in order to scale these results to submillimeter wavelengths, certain device properties should be scaled. The tunnel-junction's current density should be increased to reduce the RC product. The device's area should be reduced to efficiently couple power from the antenna to the mixer. Finally, the superconductor used should have a large energy gap to minimize RF losses. Most SIS mixers use Nb or Pb-alloy tunnel junctions; the gap frequency for these materials is approximately 725 GHz. Above the gap frequency, these materials exhibit losses similar to those in a normal metal. The gap frequency in NbN films is as-large-as 1440 GHz. Therefore, we have developed a process to fabricate small area (down to 0.13 sq microns), high current density, NbN/MgO/NbN tunnel junctions.

  1. Re-establishment of gap junctional intercellular communication (GJIC) between human endometrial carcinomas by prostaglandin E(2).

    PubMed

    Schlemmer, Scott R; Kaufman, David G

    2012-12-01

    Reduced intercellular communication via gap junctions is correlated with carcinogenesis. Gap junctional intercellular communication (GJIC), between normal human endometrial epithelial cells is enhanced when endometrial stromal cells were present in culture. This enhancement of GJIC between normal epithelial cells also occurs when they are cultured in medium conditioned by stromal cells. This observation indicated that a soluble compound (or compounds) produced and secreted by stromal cells mediates GJIC in epithelial cells. Previous studies have shown that endometrial stromal cells release prostaglandin E(2) (PGE(2)) and prostaglandin F(2α) (PGF(2α)) under physiological conditions. When we evaluated the response of normal endometrial epithelial cells to various concentrations of PGE(2,) we found enhanced GJIC with 1nM PGE(2). This is a smaller increase in GJIC than that induced by medium conditioned by stromal cells. When the extracellular concentration of PGE(2) was measured after incubation with stromal cells, it was found to be similar to the concentrations showing maximal GJIC between the normal epithelial cells. When indomethacin was used to inhibit prostaglandin synthesis by stromal cells, GJIC was reduced but not eliminated between normal endometrial epithelial cells. These observations suggest that although PGE(2) secreted by stromal cells is an important mediator of GJIC between the epithelial cells, it is not the sole mediator. Transformed endometrial epithelial cells did not demonstrate GJIC even in the presence of stromal cells. However, we were able to re-establish GJIC in transformed epithelial cells when we added PGE(2) to the cells. Our findings show that PGE(2) may serve as an intercellular mediator between stromal and epithelial cells that regulates GJIC in normal and malignant epithelial cells. This suggests that maintenance of GJIC by preserving or replacing PGE(2) secretion by endometrial stromal cells may have the potential to suppress

  2. Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

    PubMed

    Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

    2016-11-01

    Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

  3. Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

    PubMed

    Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

    2015-07-10

    Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Electrokinetic mixing vortices due to electrolyte depletion at microchannel junctions.

    PubMed

    Takhistov, Paul; Duginova, Ksenia; Chang, Hsueh-Chia

    2003-07-01

    Due to electric field leakage across sharp corners, the irrotational character of Ohmic electroosmotic flow is violated. Instead, we demonstrate experimentally and theoretically evidence of electrolyte depletion and vortex separation in electroosmotic flow around a junction between wide and narrow channels. When the penetration length of the electric field exceeds the width of the narrow channel and if the electric field is directed from the narrow to the wide channel, the electromigration of ions diminishes significantly at the junction end of the narrow channel due to this leakage. Concentration depletion then develops at that location to maintain current balance but it also increases the corner zeta potential and the local electroosmotic slip velocity. A back pressure gradient hence appears to maintain flow balance and, at a sufficient magnitude, generates a pair of vortices.

  5. Monocytic cell junction proteins serve important roles in atherosclerosis via the endoglin pathway

    PubMed Central

    Chen, Lina; Chen, Zhongliang; Ge, Menghua; Tang, Oushan; Cheng, Yinhong; Zhou, Haoliang; Shen, Yu; Qin, Fengming

    2017-01-01

    The formation of atherosclerosis is recognized to be caused by multiple factors including pathogenesis in monocytes during inflammation. The current study provided evidence that monocytic junctions were significantly altered in patients with atherosclerosis, which suggested an association between cell junctions and atherosclerosis. Claudin-1, occludin-1 and ZO-1 were significantly enhanced in atherosclerosis, indicating that the tight junction pathway was activated during the pathogenesis of atherosclerosis. In addition, the gene expression of 5 connexin members involved in the gap junction pathway were quantified, indicating that connexin 43 and 46 were significantly up-regulated in atherosclerosis. Furthermore, inflammatory factors including endoglin and SMAD were observed, suggesting that immune regulative factors were down-regulated in this pathway. Silicon-based analysis additionally identified that connexins and tight junctions were altered in association with monocytic inflammation regulations, endoglin pathway. The results imply that reduced expression of the immune regulation pathway in monocytes is correlated with the generation of gap junctions and tight junctions which serve important roles in atherosclerosis. PMID:28901429

  6. Mechanics of flow and sediment transport in delta distributary channels

    USGS Publications Warehouse

    Nelson, Jonathan M.; Kinzel, Paul J.; Duc Toan, Duong; Shimizu, Yasuyuki; McDonald, Richard R.

    2011-01-01

    boundary conditions. Over time, the pattern of erosion and deposition in the distributary channels gives rise to variations in the amount of water and sediment routed into them. In the simplest case, this results in channel switching on deltas, but in a more general sense these dynamics produce a rich suite of interesting morphologic change contributing both to the evolution of the channel distributary network and the overall evolution of the delta. As part of a study to develop a better understanding of these processes, we conducted a field study measuring the detailed morphology of the Hong-Luoc junction on the Red River downstream of Hanoi, Vietnam. This junction was selected for such a study because it has a 1,000-year history, modern observations suggest that it is currently switching (changing the proportion of sediment and streamflow provided to each of the distributary channels), and hydrologic configuration of the junction allows for the study of two bifurcations and one confluence in a single junction complex. In this paper, our morphologic observations are used in computational flow models to show how flow and sediment transport changes as a function of total discharge upstream of the junction. This is a key component of understanding how the junction attains stability over a range of flows or how imbalances in the distribution of flow and sediment transport lead to destabilization of the channel bifurcation.

  7. Gap junctions contribute to anchorage-independent clustering of breast cancer cells.

    PubMed

    Gava, Fabien; Rigal, Lise; Mondesert, Odile; Pesce, Elise; Ducommun, Bernard; Lobjois, Valérie

    2018-02-27

    Cancer cell aggregation is a key process involved in the formation of clusters of circulating tumor cells. We previously reported that cell-cell adhesion proteins, such as E-cadherin, and desmosomal proteins are involved in cell aggregation to form clusters independently of cell migration or matrix adhesion. Here, we investigated the involvement of gap junction intercellular communication (GJIC) during anchorage-independent clustering of MCF7 breast adenocarcinoma cells. We used live cell image acquisition and analysis to monitor the kinetics of MCF7 cell clustering in the presence/absence of GJIC pharmacological inhibitors and to screen a LOPAC® bioactive compound library. We also used a calcein transfer assay and flow cytometry to evaluate GJIC involvement in cancer cell clustering. We first demonstrated that functional GJIC are established in the early phase of cancer cell aggregation. We then showed that pharmacological inhibition of GJIC using tonabersat and meclofenamate delayed MCF7 cell clustering and reduced calcein transfer. We also found that brefeldin A, an inhibitor of vesicular trafficking, which we identified by screening a small compound library, and latrunculin A, an actin cytoskeleton-disrupting agent, both impaired MCF7 cell clustering and calcein transfer. Our results demonstrate that GJIC are involved from the earliest stages of anchorage-independent cancer cell aggregation. They also give insights into the regulatory mechanisms that could modulate the formation of clusters of circulating tumor cells.

  8. Associations of chemo- and radio-resistant phenotypes with the gap junction, adhesion and extracellular matrix in a three-dimensional culture model of soft sarcoma.

    PubMed

    Bai, Chujie; Yang, Min; Fan, Zhengfu; Li, Shu; Gao, Tian; Fang, Zhiwei

    2015-06-10

    Three-dimensional (3D) culture models are considered to recapitulate the cell microenvironment in solid tumors, including the extracellular matrix (ECM), cell-cell interactions, and signal transduction. These functions are highly correlated with cellular behaviors and contribute to resistances against chemo- and radio-therapies. However, the biochemical effects and mechanisms remain unknown in soft sarcoma. Therefore, we developed an in vitro 3D model of sarcoma to analyze the reasons of the chemo- and radio-resistance in therapies. Four soft sarcoma cell lines, HT1080, RD, SW872, and human osteosarcoma cell line 1 (HOSS1), a cell line established from a patient-derived xenograft, were applied to 3D culture and treated with growth factors in methylcellulose-containing medium. Spheroids were examined morphologically and by western blotting, RT-qPCR, and immunofluorescence staining to analyze cell adhesion, gap junctions, ECM genes, and related factors. Proliferation and colony formation assays were performed to assess chemo- and radio-resistances between 3D and two-dimensional (2D) cell cultures. Annexin V and Propidium Iodide staining was used to detect early apoptotic sarcoma cells treated with Doxorubicin, Gemcitabine, and Docetaxel in the 3D model. The four soft sarcoma cell lines formed spheres in vitro by culture in modified condition medium. Compared with 2D cell culture, expression of ECM genes and proteins, including COL1A1, LOX, SED1, FN1, and LAMA4, was significantly increased in 3D culture. Analysis of cadherin and gap junction molecules showed significant changes in the gene and protein expression profiles under 3D conditions. These changes affected cell-cell communication and were mainly associated with biological processes such as cell proliferation and apoptosis related to chemo- and radio-resistances. Our findings revealed significant differences between 3D and 2D cell culture systems, and indicated that cellular responsiveness to external stress

  9. Relaxation process of the discharge channel near the anode in long air gaps under positive impulse voltages

    NASA Astrophysics Data System (ADS)

    Zhao, Xiangen; He, Junjia; Luo, Bing; Jia, Lei; Yang, Yongchao; Xiao, Pei

    2017-12-01

    The relaxation process of the discharge channel near the anode in a long air gap was observed using a Schlieren system with a temporal resolution of 5 µs and a spatial resolution of 70 µm. The dynamic characteristics of the decay process in the vicinity of the anode are obtained. The discharge channel evolves just as a growing mushroom in nature during the relaxation phase. Two physical quantities, angle θ and velocity v, are defined to describe the process in this paper. The average value of the angle and velocity under lightning impulses are 71.7° and 3.3 m s-1 respectively, while 7.7 m s-1 under switching impulses. A simplified model was established to simulate the formation of mushroom-shaped channel. The simulation and experimental results show that the formation and development of the mushroom-shaped channel are due to two factors. One is the convection of the high temperature and high pressure air near the anode produced by the first corona discharge; the other is the ionic migration. These two factors result in the phenomena that the cooling process in the vicinity of the anode is much more efficient than further into the gap, whereas the thermal conductivity of the anode may have little contribution to that.

  10. Graded junction termination extensions for electronic devices

    NASA Technical Reports Server (NTRS)

    Merrett, J. Neil (Inventor); Isaacs-Smith, Tamara (Inventor); Sheridan, David C. (Inventor); Williams, John R. (Inventor)

    2006-01-01

    A graded junction termination extension in a silicon carbide (SiC) semiconductor device and method of its fabrication using ion implementation techniques is provided for high power devices. The properties of silicon carbide (SiC) make this wide band gap semiconductor a promising material for high power devices. This potential is demonstrated in various devices such as p-n diodes, Schottky diodes, bipolar junction transistors, thyristors, etc. These devices require adequate and affordable termination techniques to reduce leakage current and increase breakdown voltage in order to maximize power handling capabilities. The graded junction termination extension disclosed is effective, self-aligned, and simplifies the implementation process.

  11. Graded junction termination extensions for electronic devices

    NASA Technical Reports Server (NTRS)

    Merrett, J. Neil (Inventor); Isaacs-Smith, Tamara (Inventor); Sheridan, David C. (Inventor); Williams, John R. (Inventor)

    2007-01-01

    A graded junction termination extension in a silicon carbide (SiC) semiconductor device and method of its fabrication using ion implementation techniques is provided for high power devices. The properties of silicon carbide (SiC) make this wide band gap semiconductor a promising material for high power devices. This potential is demonstrated in various devices such as p-n diodes, Schottky diodes, bipolar junction transistors, thyristors, etc. These devices require adequate and affordable termination techniques to reduce leakage current and increase breakdown voltage in order to maximize power handling capabilities. The graded junction termination extension disclosed is effective, self-aligned, and simplifies the implementation process.

  12. Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila.

    PubMed

    Ayukawa, Tomonori; Matsumoto, Kenjiroo; Ishikawa, Hiroyuki O; Ishio, Akira; Yamakawa, Tomoko; Aoyama, Naoki; Suzuki, Takuya; Matsuno, Kenji

    2012-09-18

    Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.

  13. Agonist-activated Ca2+ influx occurs at stable plasma membrane and endoplasmic reticulum junctions

    PubMed Central

    Treves, Susan; Vukcevic, Mirko; Griesser, Johanna; Armstrong, Clara-Franzini; Zhu, Michael X.; Zorzato, Fancesco

    2010-01-01

    Junctate is a 33 kDa integral protein of sarco(endo)plasmic reticulum membranes that forms a macromolecular complex with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and TRPC3 channels. TIRF microscopy shows that junctate enhances the number of fluorescent puncta on the plasma membrane. The size and distribution of these puncta are not affected by the addition of agonists that mobilize Ca2+ from Ins(1,4,5)P3-sensitive stores. Puncta are associated with a significantly larger number of peripheral junctions between endoplasmic reticulum and plasma membrane, which are further enhanced upon stable co-expression of junctate and TRPC3. The gap between the membranes of peripheral junctions is bridged by regularly spaced electron-dense structures of 10 nm. Ins(1,4,5)P3 inhibits the interaction of the cytoplasmic N-terminus of junctate with the ligand-binding domain of the Ins(1,4,5)P3 receptor. Furthermore, Ca2+ influx evoked by activation of Ins(1,4,5)P3 receptors is increased where puncta are located. We conclude that stable peripheral junctions between the plasma membrane and endoplasmic reticulum are the anatomical sites of agonist-activated Ca2+ entry. PMID:21062895

  14. GdN nanoisland-based GaN tunnel junctions.

    PubMed

    Krishnamoorthy, Sriram; Kent, Thomas F; Yang, Jing; Park, Pil Sung; Myers, Roberto C; Rajan, Siddharth

    2013-06-12

    Tunnel junctions could have a great impact on gallium nitride and aluminum nitride-based devices such as light-emitting diodes and lasers by overcoming critical challenges related to hole injection and p-contacts. This paper demonstrates the use of GdN nanoislands to enhance interband tunneling and hole injection into GaN p-n junctions by several orders of magnitude, resulting in low tunnel junction specific resistivity (1.3 × 10(-3) Ω-cm(2)) compared to the previous results in wide band gap semiconductors. Tunnel injection of holes was confirmed by low-temperature operation of GaN p-n junction with a tunneling contact layer, and strong electroluminescence down to 20 K. The low tunnel junction resistance combined with low optical absorption loss in GdN is very promising for incorporation in GaN-based light emitters.

  15. Imaging snake orbits at graphene n -p junctions

    NASA Astrophysics Data System (ADS)

    Kolasiński, K.; Mreńca-Kolasińska, A.; Szafran, B.

    2017-01-01

    We consider conductance mapping of the snake orbits confined along the n -p junction defined in graphene by the electrostatic doping in the quantum Hall regime. We explain the periodicity of conductance oscillations at the magnetic field and the Fermi energy scales by the properties of the n -p junction as a conducting channel. We evaluate the conductance maps for a floating gate scanning the surface of the device. In the quantum Hall conditions the currents flow near the edges of the sample and along the n -p junction. The conductance mapping resolves only the n -p junction and not the edges. The conductance oscillations along the junction are found in the maps with periodicity related to the cyclotron orbits of the scattering current. Stronger probe potentials provide support to localized resonances at one of the sides of the junction with current loops that interfere with the n -p junction currents. The interference results in a series of narrow lines parallel to the junction with positions that strongly depend on the magnetic field through the Aharonov-Bohm effect. The consequences of a limited transparency of finite-width n -p junctions are also discussed.

  16. Multi-channel unidirectional transmission of phononic crystal heterojunctions

    NASA Astrophysics Data System (ADS)

    Xu, Zhenlong; Tong, Jie; Wu, Fugen

    2018-02-01

    Two square steel columns are arranged in air to form two-dimensional square lattice phononic crystals (PNCs). Two PNCs can be combined into a non-orthogonal 45∘ heterojunction when the difference in the directional band gaps of the two PNC types is utilized. The finite element method is used to calculate the acoustic band structure, the heterogeneous junction transmission characteristics, acoustic field distribution, and many others. Results show that a non-orthogonal PNC heterojunction can produce a multi-channel unidirectional transmission of acoustic waves. With the square scatterer rotated, the heterojunction can select a frequency band for unidirectional transmission performance. This capability is particularly useful for constructing acoustic diodes with wide-bands and high-efficiency unidirectional transmission characteristics.

  17. Remodelling of cellular excitation (reaction) and intercellular coupling (diffusion) by chronic atrial fibrillation represented by a reaction-diffusion system

    NASA Astrophysics Data System (ADS)

    Zhang, Henggui; Garratt, Clifford J.; Kharche, Sanjay; Holden, Arun V.

    2009-06-01

    Human atrial tissue is an excitable system, in which myocytes are excitable elements, and cell-to-cell electrotonic interactions are via diffusive interactions of cell membrane potentials. We developed a family of excitable system models for human atrium at cellular, tissue and anatomical levels for both normal and chronic atrial fibrillation (AF) conditions. The effects of AF-induced remodelling of cell membrane ionic channels (reaction kinetics) and intercellular gap junctional coupling (diffusion) on atrial excitability, conduction of excitation waves and dynamics of re-entrant excitation waves are quantified. Both ionic channel and gap junctional coupling remodelling have rate dependent effects on atrial propagation. Membrane channel conductance remodelling allows the propagation of activity at higher rates than those sustained in normal tissue or in tissue with gap junctional remodelling alone. Membrane channel conductance remodelling is essential for the propagation of activity at rates higher than 300/min as seen in AF. Spatially heterogeneous gap junction coupling remodelling increased the risk of conduction block, an essential factor for the genesis of re-entry. In 2D and 3D anatomical models, the dynamical behaviours of re-entrant excitation waves are also altered by membrane channel modelling. This study provides insights to understand the pro-arrhythmic effects of AF-induced reaction and diffusion remodelling in atrial tissue.

  18. Comparative 1D and 3D numerical investigation of open-channel junction flows and energy losses

    NASA Astrophysics Data System (ADS)

    Luo, Hao; Fytanidis, Dimitrios K.; Schmidt, Arthur R.; García, Marcelo H.

    2018-07-01

    The complexity of open channel confluences stems from flow mixing, secondary circulation, post-confluence flow separation, contraction and backwater effects. These effects in turn result in a large number of parameters required to adequately quantify the junction induced hydraulic resistance and describe mean flow pattern and turbulent flow structures due to flow merging. The recent development in computing power advances the application of 3D Computational Fluid Dynamics (CFD) codes to visualize and understand the Confluence Hydrodynamic Zone (CHZ). Nevertheless, 1D approaches remain the mainstay in large drainage network or waterway system modeling considering computational efficiency and data availability. This paper presents (i) a modified 1D nonlinear dynamic model; (ii) a fully 3D non-hydrostatic, Reynolds-averaged Navier-Stokes Equations (RANS)-based, Computational Fluid Dynamics (CFD) model; (iii) an analysis of changing confluence hydrodynamics and 3D turbulent flow structure under various controls; (iv) a comparison of flow features (i.e. upstream water depths, energy losses and post-confluence contraction) predicted by 1D and 3D models; and (v) parameterization of 3D flow characteristics in 1D modeling through the computation of correction coefficients associated with contraction, energy and momentum. The present comprehensive 3D numerical investigation highlights the driving mechanisms for junction induced energy losses. Moreover, the comparative 1D and 3D study quantifies the deviation of 1D approximations and associated underlying assumptions from the 'true' resultant flow field. The study may also shed light on improving the accuracy of the 1D large network modeling through the parameterization of the complex 3D feature of the flow field and correction of interior boundary conditions at junctions of larger angles and/or with substantial lateral inflows. Moreover, the enclosed numerical investigations may enhance the understanding of the primary

  19. Electromechanical resistive switching via back-to-back Schottky junctions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Lijie, E-mail: L.Li@swansea.ac.uk

    The physics of the electromechanical resistive switching is uncovered using the theory of back-to-back Schottky junctions combined with the quantum domain space charge transport. A theoretical model of the basic element of resistive switching devices realized by the metal-ZnO nanowires-metal structure has been created and analyzed. Simulation results show that the reverse biased Schottky junction and the air gap impedance dominate the current-voltage relation at higher external voltages; thereby electromechanically varying the air gap thickness causes the device exhibit resistive tuning characteristics. As the device dimension is in nanometre scale, investigation of the model based on quantum mechanics has alsomore » been conducted.« less

  20. Method and apparatus for controlling cross contamination of microfluid channels

    DOEpatents

    Hasselbrink, Jr., Ernest F.; Rehm, Jason E [Alameda, CA; Paul, Phillip H [Livermore, CA; Arnold, Don W [Livermore, CA

    2006-02-07

    A method for controlling fluid flow at junctions in microchannel systems. Control of fluid flow is accomplished generally by providing increased resistance to electric-field and pressure-driven flow in the form of regions of reduced effective cross-sectional area within the microchannels and proximate a channel junction. By controlling these flows in the region of a microchannel junction it is possible to eliminate sample dispersion and cross contamination and inject well-defined volumes of fluid from one channel to another.

  1. Many-junction photovoltaic device performance under non-uniform high-concentration illumination

    NASA Astrophysics Data System (ADS)

    Valdivia, Christopher E.; Wilkins, Matthew M.; Chahal, Sanmeet S.; Proulx, Francine; Provost, Philippe-Olivier; Masson, Denis P.; Fafard, Simon; Hinzer, Karin

    2017-09-01

    A parameterized 3D distributed circuit model was developed to calculate the performance of III-V solar cells and photonic power converters (PPC) with a variable number of epitaxial vertically-stacked pn junctions. PPC devices are designed with many pn junctions to realize higher voltages and to operate under non-uniform illumination profiles from a laser or LED. Performance impacts of non-uniform illumination were greatly reduced with increasing number of junctions, with simulations comparing PPC devices with 3 to 20 junctions. Experimental results using Azastra Opto's 12- and 20-junction PPC illuminated by an 845 nm diode laser show high performance even with a small gap between the PPC and optical fiber output, until the local tunnel junction limit is reached.

  2. Mechanotransductive Regulation of Gap-Junction Activity Between MLO-Y4 Osteocyte-Like and MC3T3-E1 Osteoblast-Like Cells in Three-Dimensional Co-Culture.

    NASA Technical Reports Server (NTRS)

    Juran, C. M.; Blaber, E. A.; Almeida, E. A. C.

    2016-01-01

    Cell and animal studies conducted onboard the International Space Station and formerly on Shuttle flights have provided groundbreaking data illuminating the deleterious biological response of bone to mechanical unloading. However the intercellular communicative mechanisms associated with the regulation of bone synthesis and bone resorption cells are still largely unknown. Connexin-43 (CX43), a gap junction protein, is hypothesized to play a significant role in osteoblast and osteocyte signaling. The purpose of this investigation was to evaluate within a novel three-dimensional microenvironment how the osteocyte-osteoblast gap-junction expression changes when cultures are exposed to exaggerated mechanical load. MLO-Y4 osteocyte-like cells were cultured on a 3D-Biotek polystyrene insert and placed in direct contact with an MC3T3-E1 pre-osteoblast co-cultured monolayer and exposed to 48 h of mechanical stimulation (pulsatile fluid flow (PFF) or monolayer cyclic stretch (MCS)) then evaluated for viability, proliferation, metabolism, and CX43 expression. Mono-cultured MLO-Y4 and MC3T3-E1 control experiments were conducted under PFF and MCS stimulation to observe how strain application stimuli (PFF cell membrane shear or MCS cell focal adhesion/attachment loading) initiates different signaling pathways or downstream regulatory controls. TotalLive cell count, viability and metabolic reduction (Trypan Blue, LIVEDead and Alamar Blue analysis respectively) indicate that mechanical activation of MC3T3-E1 cells inhibits proliferation while maintaining an average 1.04E4 reductioncell metabolic rate, *p0.05 n4. MLO-Y4s in monolayer culture increase in number when exposed to MCS loading but the percent of live cells within the population is low (46.3 total count, *p0.05 n4), these results may indicate an apoptotic signaling cascade. PFF stimulation of the three-dimensional co-cultures elicits a universal increase in CX43 in MLO-Y4 and MC3T3-E1 cells, illustrated by

  3. IJS: An Intelligent Junction Selection Based Routing Protocol for VANET to Support ITS Services.

    PubMed

    Bhoi, Sourav Kumar; Khilar, Pabitra Mohan

    2014-01-01

    Selecting junctions intelligently for data transmission provides better intelligent transportation system (ITS) services. The main problem in vehicular communication is high disturbances of link connectivity due to mobility and less density of vehicles. If link conditions are predicted earlier, then there is a less chance of performance degradation. In this paper, an intelligent junction selection based routing protocol (IJS) is proposed to transmit the data in a quickest path, in which the vehicles are mostly connected and have less link connectivity problem. In this protocol, a helping vehicle is set at every junction to control the communication by predicting link failures or network gaps in a route. Helping vehicle at the junction produces a score for every neighboring junction to forward the data to the destination by considering the current traffic information and selects that junction which has minimum score. IJS protocol is implemented and compared with GyTAR, A-STAR, and GSR routing protocols. Simulation results show that IJS performs better in terms of average end-to-end delay, network gap encounter, and number of hops.

  4. IJS: An Intelligent Junction Selection Based Routing Protocol for VANET to Support ITS Services

    PubMed Central

    Khilar, Pabitra Mohan

    2014-01-01

    Selecting junctions intelligently for data transmission provides better intelligent transportation system (ITS) services. The main problem in vehicular communication is high disturbances of link connectivity due to mobility and less density of vehicles. If link conditions are predicted earlier, then there is a less chance of performance degradation. In this paper, an intelligent junction selection based routing protocol (IJS) is proposed to transmit the data in a quickest path, in which the vehicles are mostly connected and have less link connectivity problem. In this protocol, a helping vehicle is set at every junction to control the communication by predicting link failures or network gaps in a route. Helping vehicle at the junction produces a score for every neighboring junction to forward the data to the destination by considering the current traffic information and selects that junction which has minimum score. IJS protocol is implemented and compared with GyTAR, A-STAR, and GSR routing protocols. Simulation results show that IJS performs better in terms of average end-to-end delay, network gap encounter, and number of hops. PMID:27433485

  5. Bipolar cell gap junctions serve major signaling pathways in the human retina.

    PubMed

    Kántor, Orsolya; Varga, Alexandra; Nitschke, Roland; Naumann, Angela; Énzsöly, Anna; Lukáts, Ákos; Szabó, Arnold; Németh, János; Völgyi, Béla

    2017-08-01

    Connexin36 (Cx36) constituent gap junctions (GJ) throughout the brain connect neurons into functional syncytia. In the retina they underlie the transmission, averaging and correlation of signals prior conveying visual information to the brain. This is the first study that describes retinal bipolar cell (BC) GJs in the human inner retina, whose function is enigmatic even in the examined animal models. Furthermore, a number of unique features (e.g. fovea, trichromacy, midget system) necessitate a reexamination of the animal model results in the human retina. Well-preserved postmortem human samples of this study are allowed to identify Cx36 expressing BCs neurochemically. Results reveal that both rod and cone pathway interneurons display strong Cx36 expression. Rod BC inputs to AII amacrine cells (AC) appear in juxtaposition to AII GJs, thus suggesting a strategic AII cell targeting by rod BCs. Cone BCs serving midget, parasol or koniocellular signaling pathways display a wealth of Cx36 expression to form homologously coupled arrays. In addition, they also establish heterologous GJ contacts to serve an exchange of information between parallel signaling streams. Interestingly, a prominent Cx36 expression was exhibited by midget system BCs that appear to maintain intimate contacts with bistratified BCs serving other pathways. These findings suggest that BC GJs in parallel signaling streams serve both an intra- and inter-pathway exchange of signals in the human retina.

  6. The role of engineered materials in superconducting tunnel junction X-ray detectors - Suppression of quasiparticle recombination losses via a phononic band gap

    NASA Technical Reports Server (NTRS)

    Rippert, Edward D.; Ketterson, John B.; Chen, Jun; Song, Shenian; Lomatch, Susanne; Maglic, Stevan R.; Thomas, Christopher; Cheida, M. A.; Ulmer, Melville P.

    1992-01-01

    An engineered structure is proposed that can alleviate quasi-particle recombination losses via the existence of a phononic band gap that overlaps the 2-Delta energy of phonons produced during recombination of quasi-particles. Attention is given to a 1D Kronig-Penny model for phonons normally incident to the layers of a multilayered superconducting tunnel junction as an idealized example. A device with a high density of Bragg resonances is identified as desirable; both Nb/Si and NbN/SiN superlattices have been produced, with the latter having generally superior performance.

  7. Old cogs, new tricks: a scaffolding role for connexin43 and a junctional role for sodium channels?

    PubMed

    Veeraraghavan, Rengasayee; Poelzing, Steven; Gourdie, Robert G

    2014-04-17

    Cardiac conduction is the process by which electrical excitation is communicated from cell to cell within the heart, triggering synchronous contraction of the myocardium. The role of conduction defects in precipitating life-threatening arrhythmias in various disease states has spurred scientific interest in the phenomenon. While the understanding of conduction has evolved greatly over the last century, the process has largely been thought to occur via movement of charge between cells via gap junctions. However, it has long been hypothesized that electrical coupling between cardiac myocytes could also occur ephaptically, without direct transfer of ions between cells. This review will focus on recent insights into cardiac myocyte intercalated disk ultrastructure and their implications for conduction research, particularly the ephaptic coupling hypothesis. Published by Elsevier B.V.

  8. Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.

    PubMed

    Milkau, Malte; Köhler, Ralf; de Wit, Cor

    2010-09-01

    Skeletal muscle activity requires substantial increases in blood flow, and the underlying vasodilation involves endothelial activity, but the contribution of the endothelium-dependent hyperpolarizing factor (EDHF) is only poorly defined. In EDHF signaling, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels SK3 and IK1 is a key step and also initiates gap junction-dependent conducted dilations. We assessed the role of SK3, IK1, and connexin40 (Cx40) in muscular contraction-induced dilations in the microcirculation in vivo. Hitherto, arterioles were observed in the electrically stimulated cremaster skeletal muscle of anesthetized mice lacking SK3, IK1, or Cx40 using intravital microscopy. Genetic deficiency of SK3, but not of IK1, strongly attenuated dilations to muscular contraction. Similarly, pharmacologic blockade of SK3 by the specific blocker UCL1684 impaired such dilations in wild-type and IK1-deficient mice. In contrast, IK1 was required for acetylcholine-induced dilations. Genetic deficiency of Cx40 also attenuated dilations induced by muscular contraction but not by acetylcholine. These data support the concept that endothelial hyperpolarization through activation of SK3 contributes to exercise hyperemia and the hyperpolarization ascends the vascular tree through gap junctions formed by Cx40 to orchestrate dilation. The differential impact of SK3- and IK1-deficiency on dilations to distinct stimuli suggests stimulus-dependent activation of these endothelial channels.

  9. Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells.

    PubMed

    Chaumontet, C; Bex, V; Gaillard-Sanchez, I; Seillan-Heberden, C; Suschetet, M; Martel, P

    1994-10-01

    Two flavones, apigenin and tangeretin, were studied for their ability to modulate gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line REL. Their cytotoxicity was first determined by cell density and neutral red uptake assays: neither apigenin nor tangeretin are cytotoxic at 10 and 25 microM, the concentrations used in our experiments. We then studied GJIC using the dye transfer assay and we observed that both apigenin and tangeretin enhance it, the maximum stimulation (x 1.7-1.8) being achieved at 25 microM for 24 h. When the dye transfer was enhanced, the amount of connexin 43 increased, which was demonstrated by Western blot and immunofluorescence analysis. For apigenin only, Northern blot analysis showed an accumulation of connexin 43 mRNA. In addition, the incubation of REL cells with the two compounds, for 1 or 24 h, prevented the inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate (1 or 10 ng/ml). The enhancement of GJIC by apigenin could be one of the major mechanisms responsible for apigenin's anti-tumour promoting action in vivo. As for tangeretin, its capacity to enhance GJIC completes its potential protective properties towards the post-initiation process.

  10. [Effect of RAAS antagonist on the expression of gap junction cx43 in myocardium of spontaneously hypertensive rat].

    PubMed

    Tan, Li-Li; Li, Lu; Liu, Li-Min; Zhao, Hong-Li

    2013-07-01

    OBJECTIVE To investigate the expression of gap junction protein Cx43 in the cardiac muscle of spontaneous hypertensive rat and the effects of various antagonists against renin angiotensin aldosterone system (RAAS) on Cx43 expression. METHODS 70 spontaneous hypertensive rats of 8-week age, 200-gram weight were separated into 7 groups, as hypertension, ramipril, telmisartan, eplerenone, ramipril + telmisartan, telmisartan + eplerenone, and ramipril+eplerenone treatment group. Another 10 healthy Wistar rats of the same age and weight were used as control group. All the rats were given intragastric administration at 8 a. m. every morning, and measured arteria caudilis pressure at 0, 4 and 8 week, respectively. 8 weeks later, all the rats were sacrificed, and the hearts were taken to measure the weight of left ventricle and the ratio of left ventricle to body weight. Myocardial fibrosis was observed by H&E staining of paraffin embedded sections, and Cx43 expression was examined by RT-PCR and western blot. The arteria caudilis pressure of spontaneous hypertensive rats was significantly higher than that of healthy control Wistar rats (P < 0.01). The decreased blood pressure was observed in RAAS antagonists treated rats, compared with hypertension group (P < 0.05). The combined treatment of telmisartan and eplerenone had the best effect of lowering blood pressure. Moreover, the weight of left ventricle, the ratio of left ventricle to body weight, myocardial fibrosis and angiotensin 11 were all prominently decreased in telmisartan and eplerenone combination group (P < 0.01). The expression of Cx43 in spontaneous hypertensive rats was significantly lower than that of healthy control Wistar rats (P < 0.01). Increased Cx43 expression was observed in RAAS antagonists treated rats, compared with hypertension group (P < 0.05). The expression of gap junction protein Cx43 was significantly down-regulated in spontaneous hypertensive rats, while RAAS antagonists increased Cx43

  11. Generation of sub-femtoliter droplet by T-junction splitting on microfluidic chips

    NASA Astrophysics Data System (ADS)

    Yang, Yu-Jun; Feng, Xuan; Xu, Na; Pang, Dai-Wen; Zhang, Zhi-Ling

    2013-03-01

    In the paper, sub-femtoliter droplets were easily produced by droplet splitting at a simple T-junction with orifice, which did not need expensive equipments, complex photolithography skill, or high energy input. The volume of the daughter droplet was not limited by channel size but controlled by channel geometry and fluidic characteristic. Moreover, single bead sampling and bead quantification in different orders of magnitude of droplet volumes were investigated. The droplets split at our T-junction chip had small volume and monodispersed size and could be produced efficiently, orderly, and controllably.

  12. Short Ballistic Josephson Coupling in Planar Graphene Junctions with Inhomogeneous Carrier Doping

    NASA Astrophysics Data System (ADS)

    Park, Jinho; Lee, Jae Hyeong; Lee, Gil-Ho; Takane, Yositake; Imura, Ken-Ichiro; Taniguchi, Takashi; Watanabe, Kenji; Lee, Hu-Jong

    2018-02-01

    We report on short ballistic (SB) Josephson coupling in junctions embedded in a planar heterostructure of graphene. Ballistic Josephson coupling is confirmed by the Fabry-Perot-type interference of the junction critical current Ic . The product of Ic and the normal-state junction resistance RN , normalized by the zero-temperature gap energy Δ0 of the superconducting electrodes, turns out to be exceptionally large close to 2, an indication of strong Josephson coupling in the SB junction limit. However, Ic shows a temperature dependence that is inconsistent with the conventional short-junction-like behavior based on the standard Kulik-Omel'yanchuk prediction. We argue that this feature stems from the effects of inhomogeneous carrier doping in graphene near the superconducting contacts, although the junction is in fact in the short-junction limit.

  13. A Comment on the Dependence of LED's Efficiency on the Junction Ideality Factor

    ERIC Educational Resources Information Center

    Sethi, Anubhav; Gupta, Yashika; Arun, P.

    2018-01-01

    P-n junctions form the basic building blocks for any semiconductor device. Therefore, the complete understanding of the junction characteristics is very important. Although being a widely discussed topic in electronics, there are still some gaps such as finding the value and significance of the junction ideality factor, that needs to be addressed.…

  14. Solution to the inverse problem of estimating gap-junctional and inhibitory conductance in inferior olive neurons from spike trains by network model simulation.

    PubMed

    Onizuka, Miho; Hoang, Huu; Kawato, Mitsuo; Tokuda, Isao T; Schweighofer, Nicolas; Katori, Yuichi; Aihara, Kazuyuki; Lang, Eric J; Toyama, Keisuke

    2013-11-01

    The inferior olive (IO) possesses synaptic glomeruli, which contain dendritic spines from neighboring neurons and presynaptic terminals, many of which are inhibitory and GABAergic. Gap junctions between the spines electrically couple neighboring neurons whereas the GABAergic synaptic terminals are thought to act to decrease the effectiveness of this coupling. Thus, the glomeruli are thought to be important for determining the oscillatory and synchronized activity displayed by IO neurons. Indeed, the tendency to display such activity patterns is enhanced or reduced by the local administration of the GABA-A receptor blocker picrotoxin (PIX) or the gap junction blocker carbenoxolone (CBX), respectively. We studied the functional roles of the glomeruli by solving the inverse problem of estimating the inhibitory (gi) and gap-junctional conductance (gc) using an IO network model. This model was built upon a prior IO network model, in which the individual neurons consisted of soma and dendritic compartments, by adding a glomerular compartment comprising electrically coupled spines that received inhibitory synapses. The model was used in the forward mode to simulate spike data under PIX and CBX conditions for comparison with experimental data consisting of multi-electrode recordings of complex spikes from arrays of Purkinje cells (complex spikes are generated in a one-to-one manner by IO spikes and thus can substitute for directly measuring IO spike activity). The spatiotemporal firing dynamics of the experimental and simulation spike data were evaluated as feature vectors, including firing rates, local variation, auto-correlogram, cross-correlogram, and minimal distance, and were contracted onto two-dimensional principal component analysis (PCA) space. gc and gi were determined as the solution to the inverse problem such that the simulation and experimental spike data were closely matched in the PCA space. The goodness of the match was confirmed by an analysis of variance

  15. Model-dependent effects of the gap junction conduction-enhancing antiarrhythmic peptide rotigaptide (ZP123) on experimental atrial fibrillation in dogs.

    PubMed

    Shiroshita-Takeshita, Akiko; Sakabe, Masao; Haugan, Ketil; Hennan, James K; Nattel, Stanley

    2007-01-23

    Abnormal intercellular communication caused by connexin dysfunction may be involved in atrial fibrillation (AF). The present study assessed the effect of the gap junctional conduction-enhancing peptide rotigaptide on AF maintenance in substrates that result from congestive heart failure induced by 2-week ventricular tachypacing (240 bpm), atrial tachypacing (ATP; 400 bpm for 3 to 6 weeks), and isolated atrial myocardial ischemia. Electrophysiological study and epicardial mapping were performed before and after rotigaptide administration in dogs with ATP and congestive heart failure, as well as in similarly instrumented sham dogs that were not tachypaced. For atrial myocardial ischemia, dogs administered rotigaptide before myocardial ischemia were compared with no-drug myocardial ischemia controls. ATP significantly shortened the atrial effective refractory period (P=0.003) and increased AF duration (P=0.008), with AF lasting >3 hours in all 6-week ATP animals. Rotigaptide increased conduction velocity in ATP dogs slightly but significantly (P=0.04) and did not affect the effective refractory period, AF duration, or atrial vulnerability. In dogs with congestive heart failure, rotigaptide also slightly increased conduction velocity (P=0.046) but failed to prevent AF promotion. Rotigaptide had no statistically significant effects in sham dogs. Myocardial ischemia alone increased AF duration and impaired conduction (based on conduction velocity across the ischemic border and indices of conduction heterogeneity). Rotigaptide prevented myocardial ischemia-induced conduction slowing and AF duration increases. Rotigaptide improves conduction in various AF models but suppresses AF only for the acute ischemia substrate. These results define the atrial antiarrhythmic profile of a mechanistically novel antiarrhythmic drug and suggest that gap junction dysfunction may be more important in ischemic AF than in ATP remodeling or congestive heart failure substrates.

  16. MYONEURAL JUNCTIONS OF TWO ULTRASTRUCTURALLY DISTINCT TYPES IN EARTHWORM BODY WALL MUSCLE

    PubMed Central

    Rosenbluth, Jack

    1972-01-01

    The longitudinal muscle of the earthworm body wall is innervated by nerve bundles containing axons of two types which form two corresponding types of myoneural junction with the muscle fibers Type I junctions resemble cholinergic neuromuscular junctions of vertebrate skeletal muscle and are characterized by three features: (a) The nerve terminals contain large numbers of spherical, clear, ∼500 A vesicles plus a small number of larger dense-cored vesicles (b) The junctional gap is relatively wide (∼900 A), and it contains a basement membrane-like material, (c) The postjunctional membrane, although not folded, displays prominent specializations on both its external and internal surfaces The cytoplasmic surface is covered by a dense matrix ∼200 A thick which appears to be the site of insertion of fine obliquely oriented cytoplasmic filaments The external surface exhibits rows of projections ∼200 A long whose bases consist of hexagonally arrayed granules seated in the outer dense layer of the plasma membrane The concentration of these hexagonally disposed elements corresponds to the estimated concentration of both receptor sites and acetylcholinesterase sites at cholinergic junctions elsewhere. Type II junctions resemble the adrenergic junctions in vertebrate smooth muscle and exhibit the following structural characteristics: (a) The nerve fibers contain predominantly dense-cored vesicles ∼1000 A in diameter (b) The junctional gap is relatively narrow (∼150 A) and contains no basement membrane-like material, (c) Postjunctional membrane specialization is minimal. It is proposed that the structural differences between the two types of myoneural junction reflect differences in the respective transmitters and corresponding differences in the mechanisms of transmitter action and/or inactivation. PMID:5044759

  17. Pronounced Photovoltaic Response from Multilayered Transition-Metal Dichalcogenides PN-Junctions.

    PubMed

    Memaran, Shahriar; Pradhan, Nihar R; Lu, Zhengguang; Rhodes, Daniel; Ludwig, Jonathan; Zhou, Qiong; Ogunsolu, Omotola; Ajayan, Pulickel M; Smirnov, Dmitry; Fernández-Domínguez, Antonio I; García-Vidal, Francisco J; Balicas, Luis

    2015-11-11

    Transition metal dichalcogenides (TMDs) are layered semiconductors with indirect band gaps comparable to Si. These compounds can be grown in large area, while their gap(s) can be tuned by changing their chemical composition or by applying a gate voltage. The experimental evidence collected so far points toward a strong interaction with light, which contrasts with the small photovoltaic efficiencies η ≤ 1% extracted from bulk crystals or exfoliated monolayers. Here, we evaluate the potential of these compounds by studying the photovoltaic response of electrostatically generated PN-junctions composed of approximately 10 atomic layers of MoSe2 stacked onto the dielectric h-BN. In addition to ideal diode-like response, we find that these junctions can yield, under AM-1.5 illumination, photovoltaic efficiencies η exceeding 14%, with fill factors of ~70%. Given the available strategies for increasing η such as gap tuning, improving the quality of the electrical contacts, or the fabrication of tandem cells, our study suggests a remarkable potential for photovoltaic applications based on TMDs.

  18. Design of thin InGaAsN(Sb) n-i-p junctions for use in four-junction concentrating photovoltaic devices

    NASA Astrophysics Data System (ADS)

    Wilkins, Matthew M.; Gupta, James; Jaouad, Abdelatif; Bouzazi, Boussairi; Fafard, Simon; Boucherif, Abderraouf; Valdivia, Christopher E.; Arès, Richard; Aimez, Vincent; Schriemer, Henry P.; Hinzer, Karin

    2017-04-01

    Four-junction solar cells for space and terrestrial applications require a junction with a band gap of ˜1 eV for optimal performance. InGaAsN or InGaAsN(Sb) dilute nitride junctions have been demonstrated for this purpose, but in achieving the 14 mA/cm2 short-circuit current needed to match typical GaInP and GaAs junctions, the open-circuit voltage (VOC) and fill factor of these junctions are compromised. In multijunction devices incorporating materials with short diffusion lengths, we study the use of thin junctions to minimize sensitivity to varying material quality and ensure adequate transmission into lower junctions. An n-i-p device with 0.65-μm absorber thickness has sufficient short-circuit current, however, it relies less heavily on field-aided collection than a device with a 1-μm absorber. Our standard cell fabrication process, which includes a rapid thermal anneal of the contacts, yields a significant improvement in diffusion length and device performance. By optimizing a four-junction cell around a smaller 1-sun short-circuit current of 12.5 mA/cm2, we produced an InGaAsN(Sb) junction with open-circuit voltage of 0.44 V at 1000 suns (1 sun=100 mW/cm2), diode ideality factor of 1.4, and sufficient light transmission to allow >12.5 mA/cm2 in all four subcells.

  19. Morphology of the cemento-enamel junction in premolar teeth.

    PubMed

    Arambawatta, Kapila; Peiris, Roshan; Nanayakkara, Deepthi

    2009-12-01

    The present study attempted to describe the distribution of the mineralized tissues that compose the cemento-enamel junction, with respect to both the different types of permanent premolars of males and females and the various surfaces of individual teeth. The cervical region of ground sections of 67 premolars that had been extracted for orthodontic reasons were analyzed using transmitted light microscopy to identify which of the following tissue interrelationships was present at the cemento-enamel junction: cementum overlapping enamel; enamel overlapping cementum; edge-to-edge relationship between cementum and enamel; or the presence of gaps between the enamel and cementum with exposed dentin. An edge-to-edge interrelation between root cementum and enamel was predominant (55.1%). In approximately one-third of the sample, gaps between cementum and enamel with exposed dentin were observed. Cementum overlapping enamel was less prevalent than previously reported, and enamel overlapping cementum was seen in a very small proportion of the sample. In any one tooth, the distribution of mineralized tissues at the cemento-enamel junction was irregular and unpredictable. The frequency of gaps between enamel and cementum with exposure of dentin was higher than previously reported, which suggests that this region is fragile and strongly predisposed to pathological changes. Hence, this region should be protected and carefully managed during routine clinical procedures such as dental bleaching, orthodontic treatment, and placement of restorative materials.

  20. A deafness mechanism of digenic Cx26 (GJB2) and Cx30 (GJB6) mutations: Reduction of endocochlear potential by impairment of heterogeneous gap junctional function in the cochlear lateral wall.

    PubMed

    Mei, Ling; Chen, Jin; Zong, Liang; Zhu, Yan; Liang, Chun; Jones, Raleigh O; Zhao, Hong-Bo

    2017-12-01

    Digenic Connexin26 (Cx26, GJB2) and Cx30 (GJB6) heterozygous mutations are the second most frequent cause of recessive deafness in humans. However, the underlying deafness mechanism remains unclear. In this study, we created different double Cx26 and Cx30 heterozygous (Cx26 +/- /Cx30 +/- ) mouse models to investigate the underlying pathological changes and deafness mechanism. We found that double Cx26 +/- /Cx30 +/- heterozygous mice had hearing loss. Endocochlear potential (EP), which is a driving force for hair cells producing auditory receptor current, was reduced. However, unlike Cx26 homozygous knockout (Cx26 -/- ) mice, the cochlea in Cx26 +/- /Cx30 +/- mice displayed normal development and had no apparent hair cell degeneration. Gap junctions (GJs) in the cochlea form two independent networks: the epithelial cell GJ network in the organ of Corti and the connective tissue GJ network in the cochlear lateral wall. We further found that double heterozygous deletion of Cx26 and Cx30 in the epithelial cells did not reduce EP and had normal hearing, suggesting that Cx26 +/- /Cx30 +/- may mainly impair gap junctional functions in the cochlear lateral wall and lead to EP reduction and hearing loss. Most of Cx26 and Cx30 in the cochlear lateral wall co-expressed in the same gap junctional plaques. Moreover, sole Cx26 +/- or Cx30 +/- heterozygous mice had no hearing loss. These data further suggest that digenic Cx26 and Cx30 mutations may impair heterozygous coupling of Cx26 and Cx30 in the cochlear lateral wall to reduce EP, thereby leading to hearing loss. Copyright © 2017 Elsevier Inc. All rights reserved.