Science.gov

Sample records for blocks gap junction

  1. Gap junctions.

    PubMed

    Goodenough, Daniel A; Paul, David L

    2009-07-01

    Gap junctions are aggregates of intercellular channels that permit direct cell-cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.

  2. Gap Junctions

    PubMed Central

    Goodenough, Daniel A.; Paul, David L.

    2009-01-01

    Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology. PMID:20066080

  3. Gap junctions.

    PubMed

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

    2012-07-01

    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 2:1853-1872, 2012.

  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. Gap junctions.

    PubMed

    Shimizu, Kazumichi; Stopfer, Mark

    2013-12-02

    In vertebrates and invertebrates, signaling among neurons is most commonly mediated by chemical synapses. At these synapses neurotransmitter released by presynaptic neurons is detected by receptors on the postsynaptic neurons, leading to an influx of ions through the receptors themselves or through channels activated by intracellular signaling downstream of the receptors. But neurons can communicate with each other in a more direct way, by passing signals composed of small molecules and ions through pores called gap junctions. Gap junctions that transmit electrical signals are called electrical synapses. Unlike most chemical synapses, electrical synapses interact through axon-to-axon or dendrite-to-dendrite contacts. Found throughout the nervous system, they are probably best known for linking the relatively few inhibitory, GABAergic, neurons into large, effective networks within vertebrate brains. They are particularly important early in development before the formation of most chemical synapses, but recent work shows gap junctions play important roles in the adult nervous system, too. Gap junctions are sometimes thought to be mere passageways between cells. But, as recent work shows, their properties can be complex and surprising. Gap junctions help generate, propagate, and regulate neural oscillations, can filter electrical signals, and can be modulated in a variety of ways. Here we discuss recent work highlighting the diversity and importance of gap junctions throughout the nervous system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells

    PubMed Central

    Li, Xin-Zhi; Ma, Ke-Tao; Guan, Bing-Cai; Li, Li; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang; Jiang, Zhi-Gen

    2013-01-01

    We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56 μM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥300 μM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62 μM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K+-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ~300 μM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K+-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300 μM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels. PMID:23420003

  7. 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.

  8. Cumulus Cells Block Oocyte Meiotic Resumption via Gap Junctions in Cumulus Oocyte Complexes Subjected to DNA Double-Strand Breaks.

    PubMed

    Sun, Ming-Hong; Zheng, Jie; Xie, Feng-Yun; Shen, Wei; Yin, Shen; Ma, Jun-Yu

    2015-01-01

    During mammalian oocyte growth, genomic DNA may accumulate DNA double-strand breaks (DSBs) induced by factors such as reactive oxygen species. Recent evidence demonstrated that slight DSBs do not activate DNA damage checkpoint proteins in denuded oocytes. These oocytes, even with DNA DSBs, can resume meiosis and progress to metaphase of meiosis II. Meiotic resumption in oocytes is also controlled by the surrounding cumulus cells; accordingly, we analyzed whether cumulus-cell enclosed oocytes (CEOs) with DNA damage are able to resume meiosis. Compared with DNA-damaged denuded oocytes, we found that meiotic resumption rates of CEOs significantly decreased. To assess the mechanism by which cumulus cells block meiotic resumption in CEOs with DNA DSBs, we treated the cumulus oocyte complex with the gap junction inhibitor carbenoxolone and found that carbenoxolone can rescue the block in CEO meiosis induced by DNA DSBs. Since cumulus cell-synthesized cAMPs can pass through the gap junctions between oocyte and cumulus cell to block oocyte meiosis, we measured the expression levels of adenylate cyclase 1 (Adcy1) in cumulus cells, and G-protein coupled receptor 3 (Gpr3) and phosphodiesterase 3A (Pde3a) in oocytes, and found that the mRNA expression level of Adcy1 increased significantly in DNA-damaged cumulus cells. In conclusion, our results indicate that DNA DSBs promote cAMP synthesis in cumulus cells, and cumulus cAMPs can inhibit meiotic resumption of CEOs through gap junctions.

  9. Gap junctions, pannexins and pain.

    PubMed

    Spray, David C; Hanani, Menachem

    2017-06-22

    Enhanced expression and function of gap junctions and pannexin (Panx) channels have been associated with both peripheral and central mechanisms of pain sensitization. At the level of the sensory ganglia, evidence includes augmented gap junction and pannexin1 expression in glial cells and neurons in inflammatory and neuropathic pain models and increased synchrony and enhanced cross-excitation among sensory neurons by gap junction-mediated coupling. In spinal cord and in suprapinal areas, evidence is largely limited to increased expression of relevant proteins, although in several rodent pain models, hypersensitivity is reduced by treatment with gap junction/Panx1 channel blocking compounds. Moreover, targeted modulation of Cx43 expression was shown to modulate pain thresholds, albeit in somewhat contradictory ways, and mice lacking Panx1 expression globally or in specific cell types show depressed hyperalgesia. We here review the evidence for involvement of gap junctions and Panx channels in a variety of animal pain studies and then discuss ways in which gap junctions and Panx channels may mediate their action in pain processing. This discussion focusses on spread of signals among satellite glial cells, in particular intercellular Ca(2+) waves, which are propagated through both gap junction and Panx1-dependent routes and have been associated with the phenomenon of spreading depression and the malady of migraine headache with aura. Copyright © 2017. Published by Elsevier B.V.

  10. Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration.

    PubMed

    Toychiev, Abduqodir H; Ivanova, Elena; Yee, Christopher W; Sagdullaev, Botir T

    2013-08-28

    Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.

  11. Gap junctions - guards of excitability.

    PubMed

    Stroemlund, Line Waring; Jensen, Christa Funch; Qvortrup, Klaus; Delmar, Mario; Nielsen, Morten Schak

    2015-06-01

    Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane.

  12. Role of gap junctions in epilepsy.

    PubMed

    Jin, Miao-Miao; Chen, Zhong

    2011-12-01

    Epilepsy is a common neurological disorder characterized by periodic and unpredictable seizures. Gap junctions have recently been proposed to be involved in the generation, synchronization and maintenance of seizure events. The present review mainly summarizes recent reports concerning the contribution of gap junctions to the pathophysiology of epilepsy, together with the regulation of connexin after clinical and experimental seizure activity. The anticonvulsant effects of gap junction blockers both in vitro and in vivo suggest that the gap junction is a candidate target for the development of antiepileptic drugs. It is also of interest that the roles of neuronal and astrocytic gap junctions in epilepsy have been investigated independently, based on evidence from pharmacological manipulations and connexin-knockout mice. Further studies using more specific manipulations of gap junctions in different cell types and in human epileptic tissue are needed to fully uncover the role of gap junctions in epilepsy.

  13. [Gap junctions and cancer: implications and perspectives].

    PubMed

    Mesnil, Marc

    2004-02-01

    Gap junctions are made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules (<1,200 Da) such as ions, sugars, amino acids, nucleotides, second messengers (calcium, inositol triphosphate, etc.). Since their discovery in the early sixties, several groups have described the loss of their function in cancer cells. The accumulation of such data led to the hypothesis that gap junctions are involved in the carcinogenesis process. This assumption has been confirmed by data establishing that gap junctional intercellular communication is inhibited by most of the tumor promoters and that the restoration of such a communication, by transfection of cDNAs encoding gap junction proteins (connexins), inhibits the aberrant growth rates of tumorigenic cells. Despite these important informations, several fundamental questions remain still open. First, we do not know how gap junctions mediate such a tumor suppressor effect and whether it may depend either on the cell type or on the connexin type. Moreover, most of the data concerning a possible involvement of gap junctions in carcinogenesis have been obtained from in vitro and animal models. The very few results which have been currently collected from human tumors are not sufficient to have a clear idea concerning the real involvement of gap junctions in sporadic human cancers. These points as well as other unresolved questions about the role of gap junctional intercellular communication in carcinogenesis are mentioned. To bring some answers, some prospects are proposed with the objective to use gap junctions for increasing the effect of anticancer therapies.

  14. In vitro formation of gap junction vesicles.

    PubMed

    Goodenough, D A

    1976-02-01

    A method is described that uses trypsin digestion combined with collagenase-hyaluronidase which produces a population of gap junction vesicles. The hexagonal lattice of subunits ("connexons") comprising the gapjunctions appears unaltered by various structural criteria and by buoyant density measurements. The gap junction vesciles are closed by either a single or a double profile of nonjunctional "membrane," which presents a smooth, particle-free fracture face. Horseradish peroxidase and cytochrome c studies have revealed that about 20% of the gap junction vesicles are impermeable to proteins 12,000 daltons or larger. The increased purity of the trypsinized junction preparation suggests that one of the disulfide reduction products of the gap-junction principal protein may be a nonjunctional contaminating peptide. The gap junction appears to be composed of a single 18,000-dalton protein, connexin, which may be reduced to a single 9,000-dalton peak. The number of peptides in this reduced peak are still unknown.

  15. Gap junctions, homeostasis, and injury.

    PubMed

    De Maio, Antonio; Vega, Virginia L; Contreras, Jorge E

    2002-06-01

    Gap junctions (Gj) play an important role in the communication between cells of many tissues. They are composed of channels that permit the passage of ions and low molecular weight metabolites between adjacent cells, without exposure to the extracellular environment. These pathways are formed by the interaction between two hemichannels on the surface of opposing cells. These hemichannels are formed by the association of six identical subunits, named connexins (Cx), which are integral membrane proteins. Cell coupling via Gj is dependent on the specific pattern of Cx gene expression. This pattern of gene expression is altered during several pathological conditions resulting in changes of cell coupling. The regulation of Cx gene expression is affected at different levels from transcription to post translational processes during injury. In addition, Gj cellular communication is regulated by gating mechanisms. The alteration of Gj communication during injury could be rationalized by two opposite theories. One hypothesis proposes that the alteration of Gj communication attenuates the spread of toxic metabolites from the injured area to healthy organ regions. The alternative proposition is that a reduction of cellular communication reduces the loss of important cellular metabolisms, such as ATP and glucose. Copyright 2002 Wiley-Liss, Inc.

  16. 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.

  17. 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

  18. 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

  19. Gap Junctions Couple Astrocytes and Oligodendrocytes

    PubMed Central

    Orthmann-Murphy, Jennifer L.; Abrams, Charles K.; Scherer, Steven S.

    2009-01-01

    In vertebrates, a family of related proteins called connexins form gap junctions (GJs), which are intercellular channels. In the central nervous system (CNS), GJs couple oligodendrocytes and astrocytes (O/A junctions) and adjacent astrocytes (A/A junctions), but not adjacent oligodendrocytes, forming a “glial syncytium.” Oligodendrocytes and astrocytes each express different connexins. Mutations of these connexin genes demonstrate that the proper functioning of myelin and oligodendrocytes requires the expression of these connexins. The physiological function of O/A and A/A junctions, however, remains to be illuminated. PMID:18236012

  20. Gap junctions: structure and function (Review).

    PubMed

    Evans, W Howard; Martin, Patricia E M

    2002-01-01

    Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to approximately 1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.

  1. Gap junctions and blood-tissue barriers.

    PubMed

    Li, Michelle W M; Mruk, Dolores D; Cheng, C Yan

    2012-01-01

    Gap junction is a cell-cell communication junction type found in virtually all mammalian epithelia and endothelia and provides the necessary "signals" to coordinate physiological events to maintain the homeostasis of an epithelium and/or endothelium under normal physiological condition and following changes in the cellular environment (e.g., stimuli from stress, growth, development, inflammation, infection). Recent studies have illustrated the significance of this junction type in the maintenance of different blood-tissue barriers, most notably the blood-brain barrier and blood-testis barrier, which are dynamic ultrastructures, undergoing restructuring in response to stimuli from the environment. In this chapter, we highlight and summarize the latest findings in the field regarding how changes at the gap junction, such as the result of a knock-out, knock-down, knock-in, or gap junction inhibition and/or its activation via the use of inhibitors and/or activators, would affect the integrity or permeability of the blood-tissue barriers. These findings illustrate that much research is needed to delineate the role of gap junction in the blood-tissue barriers, most notably its likely physiological role in mediating or regulating the transport of therapeutic drugs across the blood-tissue barriers.

  2. GAP JUNCTIONS AND BLOOD-TISSUE BARRIERS

    PubMed Central

    Li, Michelle W.M.; Mruk, Dolores D.; Cheng, C. Yan

    2014-01-01

    Gap junction is a cell-cell communication junction type found in virtually all mammalian epithelia and endothelia and provides the necessary “signals” to coordinate physiological events to maintain the homeostasis of an epithelium and/or endothelium under normal physiological condition and following changes in the cellular environment (e.g., stimuli from stress, growth, development, inflammation, infection). Recent studies have illustrated the significance of this junction type in the maintenance of different blood-tissue barriers, most notably the blood-brain barrier and blood-testis barrier, which are dynamic ultrastructures, undergoing restructuring in response to stimuli from the environment. In this chapter, we highlight and summarize the latest findings in the field regarding how changes at the gap junction, such as the result of a knock-out, knock-down, knock-in, or gap junction inhibition and/or its activation via the use of inhibitors and/or activators, would affect the integrity or permeability of the blood-tissue barriers. These findings illustrate that much research is needed to delineate the role of gap junction in the blood-tissue barriers, most notably its likely physiological role in mediating or regulating the transport of therapeutic drugs across the blood-tissue barriers. PMID:23397629

  3. 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.

  4. Roles of gap junctions, connexins, and pannexins in epilepsy

    PubMed Central

    Mylvaganam, Shanthini; Ramani, Meera; Krawczyk, Michal; Carlen, Peter L.

    2014-01-01

    Enhanced gap junctional communication (GJC) between neurons is considered a major factor underlying the neuronal synchrony driving seizure activity. In addition, the hippocampal sharp wave ripple complexes, associated with learning and seizures, are diminished by GJC blocking agents. Although gap junctional blocking drugs inhibit experimental seizures, they all have other non-specific actions. Besides interneuronal GJC between dendrites, inter-axonal and inter-glial GJC is also considered important for seizure generation. Interestingly, in most studies of cerebral tissue from animal seizure models and from human patients with epilepsy, there is up-regulation of glial, but not neuronal gap junctional mRNA and protein. Significant changes in the expression and post-translational modification of the astrocytic connexin Cx43, and Panx1 were observed in an in vitro Co++ seizure model, further supporting a role for glia in seizure-genesis, although the reasons for this remain unclear. Further suggesting an involvement of astrocytic GJC in epilepsy, is the fact that the expression of astrocytic Cx mRNAs (Cxs 30 and 43) is several fold higher than that of neuronal Cx mRNAs (Cxs 36 and 45), and the number of glial cells outnumber neuronal cells in mammalian hippocampal and cortical tissue. Pannexin expression is also increased in both animal and human epileptic tissues. Specific Cx43 mimetic peptides, Gap 27 and SLS, inhibit the docking of astrocytic connexin Cx43 proteins from forming intercellular gap junctions (GJs), diminishing spontaneous seizures. Besides GJs, Cx membrane hemichannels in glia and Panx membrane channels in neurons and glia are also inhibited by traditional gap junctional pharmacological blockers. Although there is no doubt that connexin-based GJs and hemichannels, and pannexin-based membrane channels are related to epilepsy, the specific details of how they are involved and how we can modulate their function for therapeutic purposes remain to be

  5. Clathrin and Cx43 gap junction plaque endoexocytosis

    SciTech Connect

    Nickel, Beth M.; DeFranco, B. Hewa; Gay, Vernon L.; Murray, Sandra A.

    2008-10-03

    In earlier transmission electron microscopic studies, we have described pentilaminar gap junctional membrane invaginations and annular gap junction vesicles coated with short, electron-dense bristles. The similarity between these electron-dense bristles and the material surrounding clathrin-coated pits led us to suggest that the dense bristles associated with gap junction structures might be clathrin. To confirm that clathrin is indeed associated with annular gap junction vesicles and gap junction plaques, quantum dot immuno-electron microscopic techniques were used. We report here that clathrin associates with both connexin 43 (Cx43) gap junction plaques and pentilaminar gap junction vesicles. An important finding was the preferential localization of clathrin to the cytoplasmic surface of the annular or of the gap junction plaque membrane of one of the two contacting cells. This is consistent with the possibility that the direction of gap junction plaque internalization into one of two contacting cells is regulated by clathrin.

  6. 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.

  7. Gap junctional communication during limb cartilage differentiation.

    PubMed

    Coelho, C N; Kosher, R A

    1991-03-01

    The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.

  8. Variety of horizontal cell gap junctions in the rabbit retina.

    PubMed

    Cha, Jiook; Kim, Hong-Lim; Pan, Feng; Chun, Myung-Hoon; Massey, Stephen C; Kim, In-Beom

    2012-02-29

    In the rabbit retina, there are two types of horizontal cell (HC). The axonless A-type HCs form a coupled network via connexin 50 (Cx50) gap junctions in the outer plexiform layer (OPL). The axon-bearing B-type HCs form two independently coupled networks; the dendritic network via gap junctions consisted of unknown Cx and the axon terminal network via Cx57. The present study was conducted to examine the localization and morphological features of Cx50 and Cx57 gap junctions in rabbit HCs at cellular and subcellular levels. The results showed that each gap junction composed of Cx50 or Cx57 showed distinct features. The larger Cx50 gap junctions were located more proximally than the smaller Cx50 gap junctions. Both Cx50 plaques formed symmetrical homotypic gap junctions, but some small ones had an asymmetrical appearance, suggesting the presence of heterotypic gap junctions or hemichannels. In contrast, Cx57 gap junctions were found in the more distal part of the OPL but never on the axon terminal endings entering the rod spherules, and they were exclusively homotypic. Interestingly, about half of the Cx57 gap junctions appeared to be invaginated. These distinct features of Cx50 and Cx57 gap junctions show the variety of HC gap junctions and may provide insights into the function of different types of HCs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. Gap junctions in several tissues share antigenic determinants with liver gap junctions.

    PubMed Central

    Dermietzel, R; Leibstein, A; Frixen, U; Janssen-Timmen, U; Traub, O; Willecke, K

    1984-01-01

    Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6209130

  10. 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.

  11. Neuro-muscular junction block stimulator simulator.

    PubMed

    Sprick, Cyle

    2006-03-01

    Improved technology and higher fidelity are making medical simulations increasingly popular. A simulated peripheral nerve stimulator and thumb actuator has been developed for use with the SimMan Universal Patient Simulator. This device incorporates a handheld control box, a McKibben pneumatic muscle and articulated thumb, and a remote software interface for the simulation facilitator. The system simulates the action of a peripheral nerve stimulator on the ulnar nerve, and the effects of neuromuscular junction blocking agents on the thumb motion.

  12. Gap Junction Dysfunction in the Prefrontal Cortex Induces Depressive-Like Behaviors in Rats

    PubMed Central

    Sun, Jian-Dong; Liu, Yan; Yuan, Yu-He; Li, Jing; Chen, Nai-Hong

    2012-01-01

    Growing evidence has implicated glial anomalies in the pathophysiology of major depression disorder (MDD). Gap junctional communication is a main determinant of astrocytic function. However, it is unclear whether gap junction dysfunction is involved in MDD development. This study investigates changes in the function of astrocyte gap junction occurring in the rat prefrontal cortex (PFC) after chronic unpredictable stress (CUS), a rodent model of depression. Animals exposed to CUS and showing behavioral deficits in sucrose preference test (SPT) and novelty suppressed feeding test (NSFT) exhibited significant decreases in diffusion of gap junction channel-permeable dye and expression of connexin 43 (Cx43), a major component of astrocyte gap junction, and abnormal gap junctional ultrastructure in the PFC. Furthermore, we analyzed the effects of typical antidepressants fluoxetine and duloxetine and glucocorticoid receptor (GR) antagonist mifepristone on CUS-induced gap junctional dysfunction and depressive-like behaviors. The cellular and behavioral alterations induced by CUS were reversed and/or blocked by treatment with typical antidepressants or mifepristone, indicating that the mechanism of their antidepressant action may involve the amelioration of gap junction dysfunction and the cellular changes may be related to GR activation. We then investigated the effects of pharmacological gap junction blockade in the PFC on depressive-like behaviors. The results demonstrate that carbenoxolone (CBX) infusions induced anhedonia in SPT, and anxiety in NSFT, and Cx43 mimetic peptides Gap27 and Gap26 also induced anhedonia, a core symptom of depression. Together, this study supports the hypothesis that gap junction dysfunction contributes to the pathophysiology of depression. PMID:22189291

  13. Expression of Functional Cell-Cell Channels from Cloned Rat Liver Gap Junction Complementary DNA

    NASA Astrophysics Data System (ADS)

    Dahl, G.; Miller, T.; Paul, D.; Voellmy, R.; Werner, R.

    1987-06-01

    An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.

  14. COEXISTENCE OF GAP AND SEPTATE JUNCTIONS IN AN INVERTEBRATE EPITHELIUM

    PubMed Central

    Hudspeth, A. J.; Revel, J. P.

    1971-01-01

    The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist. PMID:5563454

  15. Structure, regulation and function of gap junctions in liver

    PubMed Central

    Maes, Michaël; Decrock, Elke; Wang, Nan; Leybaert, Luc; da Silva, Tereza Cristina; Veloso Alves Pereira, Isabel; Jaeschke, Hartmut; Cogliati, Bruno; Vinken, Mathieu

    2016-01-01

    Gap junctions are a specialized group of cell-to-cell junctions that mediate direct intercellular communication between cells. They arise from the interaction of 2 hemichannels of adjacent cells, which in turn are composed of 6 connexin proteins. In liver, gap junctions are predominantly found in hepatocytes and play critical roles in virtually all phases of the hepatic life cycle, including cell growth, differentiation, liver-specific functionality and cell death. Liver gap junctions are directed through a broad variety of mechanisms ranging from epigenetic control of connexin expression to posttranslational regulation of gap junction activity. This paper reviews established and novel aspects regarding the architecture, control and functional relevance of liver gap junctions. PMID:27001459

  16. Structure, Regulation and Function of Gap Junctions in Liver.

    PubMed

    Willebrords, Joost; Crespo Yanguas, Sara; Maes, Michaël; Decrock, Elke; Wang, Nan; Leybaert, Luc; da Silva, Tereza Cristina; Veloso Alves Pereira, Isabel; Jaeschke, Hartmut; Cogliati, Bruno; Vinken, Mathieu

    Gap junctions are a specialized group of cell-to-cell junctions that mediate direct intercellular communication between cells. They arise from the interaction of two hemichannels of adjacent cells, which in turn are composed of six connexin proteins. In liver, gap junctions are predominantly found in hepatocytes and play critical roles in virtually all phases of the hepatic life cycle, including cell growth, differentiation, liver-specific functionality and cell death. Liver gap junctions are directed through a broad variety of mechanisms ranging from epigenetic control of connexin expression to post-translational regulation of gap junction activity. This paper reviews established and novel aspects regarding the architecture, control and functional relevance of liver gap junctions.

  17. 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...

  18. 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...

  19. 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.

  20. 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.

  1. Glial Connexins and Gap Junctions in CNS inflammation and disease

    PubMed Central

    Kielian, Tammy

    2009-01-01

    Gap junctions facilitate direct cytoplasmic communication between neighboring cells, facilitating the transfer of small molecular weight molecules involved in cell signaling and metabolism. Gap junction channels are formed by the joining of two hemichannels from adjacent cells, each composed of six oligomeric protein subunits called connexins (Cx). Of paramount importance to CNS homeostasis are astrocyte networks formed by gap junctions, which play a critical role in maintaining the homeostatic regulation of extracellular pH, K+, and glutamate levels. Inflammation is a hallmark of several diseases afflicting the CNS. Within the past several years, the number of publications reporting effects of cytokines and pathogenic stimuli on glial gap junction communication has increased dramatically. The purpose of this review is to discuss recent observations characterizing the consequences of inflammatory stimuli on homocellular gap junction coupling in astrocytes and microglia as well as changes in connexin expression during various CNS inflammatory conditions. PMID:18410504

  2. Gap junctional conductance and permeability are linearly related.

    PubMed

    Verselis, V; White, R L; Spray, D C; Bennett, M V

    1986-10-24

    The permeability of gap junctions to tetraethylammonium ions was measured in isolated pairs of blastomeres from Rana pipiens L. and compared to the junctional conductance. In this system, the junctional conductance is voltage-dependent and decreases with moderate transjunctional voltage of either sign. The permeability to tetraethylammonium ions was determined by injecting one cell of a pair with tetraethylammonium and monitoring its changing concentration in the prejunctional and postjunctional cells with ion-selective electrodes. Junctional conductance was determined by current-clamp and voltage-clamp techniques. For different cell pairs in which the transjunctional voltage was small and the junctional conductance at its maximum value, the permeability to tetraethylammonium ions was proportional to the junctional conductance. In individual cell pairs, a reduction in the junctional conductance induced by voltage was accompanied by a proportional reduction in the permeability of the gap junction over a wide range. The diameter of the tetraethylammonium ion (8.0 to 8.5 A, unhydrated) is larger than that of the potassium ion (4.6 A, hydrated), the predominant current-carrying species. The proportionality between the permeability to tetraethylammonium ions and the junctional conductance, measured here with exceptionally fine time resolution, indicates that a common gap junctional pathway mediates both electrical and chemical fluxes between cells, and that closure of single gap junction channels by voltage is all or none.

  3. Comparative analysis of the gap junction protein from rat heart and liver: is there a tissue specificity of gap junctions?

    PubMed

    Gros, D B; Nicholson, B J; Revel, J P

    1983-12-01

    Gap junctions have been isolated from both rat heart and liver, tissues where junctions are typical in appearance and physiology. The purity of the fractions obtained was monitored by electron microscopy (thin-sectioning and negative staining) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The myocardial gap junctions are comprised of a single polypeptide of Mr 28,000, apparently derived from a protein of Mr 30,000. Hepatic gap junctions are also comprised of a single native protein of Mr 28,000 as previously reported. Exhaustive trypsin digestion of the isolated junctions cleaves both of these proteins similarly, while leaving their characteristic junctional lattice structures intact. However, comparison of heart and liver junctional proteins by two-dimensional peptide mapping of tryptic and alpha-chymotryptic fragments, followed by high pressure liquid chromatography, reveals no homology between these proteins.

  4. 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

  5. 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

  6. Regulation of gap junctional communication during human trophoblast differentiation.

    PubMed

    Cronier, L; Hervé, J C; Délèze, J; Malassiné, A

    During pregnancy, the trophoblast, supporting the main functions of the placenta, develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. Gap junction channels consisting of connexins link the cytosols of cells in contact. Gap junctional communication has been involved in the control of cell and tissue differentiation. Recently, a gap junctional communication was demonstrated in trophoblast cell culture by means of the fluorescence recovery after photobleaching (gap-FRAP) technique. This gap junctional communication appeared to be stimulated by human chorionic gonadotropin (hCG). Therefore, the specificity of hCG action and the signalling mechanisms implicated in gap junctional communication were investigated by means of gap-FRAP. In culture, cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 1-2 days after plating. During this in vitro differentiation, gap junctional communication was measured, and the maximum percentage of coupling between adjacent cells occurred on the fourth day. In the presence of 500 mIU/ml hCG, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days instead of 4 days in control conditions. The hCG action was specific, since the addition of heat-inactivated hCG of oFSH or of bTSH did not affect gap junctional communication in trophoblastic cells. The addition of a polyclonal hCG antibody decreased basal gap junctional communication as well as the response to exogenous hCG. Moreover, the presence of 8Br-cAMP (0.5 or 1 mM) mimicked the stimulation by hCG. Interestingly, H89 (2 microM), a specific protein kinase-A inhibitor, dramatically decreased the responses to hCG (500 mIU/ml) and the 8Br-cAMP (0.5 mM) stimulation of trophoblastic gap junctional communication. Calphostin (1 or 2 microM), a specific protein kinase-C inhibitor, strongly stimulated gap junctional communication. In conclusion, the

  7. 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.

  8. Gap junction channels reconstituted in two closely apposed lipid bilayers.

    PubMed

    Ramundo-Orlando, Alfonsina; Serafino, Annalucia; Villalobo, Antonio

    2005-04-01

    Intercellular communication mediated by gap junction channels plays an important role in many cellular processes. In contrast to other channels, gap junction channels span two plasma membranes resulting in an intracellular location for both ends of the junctional pore and the regulatory sites for channel gating. This configuration presents unique challenges for detailed experimental studies of junctional channel physiology and ligand-activation in situ. Availability of an appropriate model system would significantly facilitate future studies of gap junction channel function and structure. Here we show that the double-membrane channel can be reconstituted in pairs of closely apposed lipid bilayers, as experienced in cells. We have trapped the calcium-sensitive dye, arsenazo III (AIII), partially calcium-saturated (AIII-Ca), in one population of connexin32 reconstituted-liposomes, and EGTA in a second one. In such mixtures, the interaction of EGTA with AIII-Ca was measured by a large color shift from blue to red (decreased absorbance at 652 nm). The exchange of these compounds through gap junctions was proportional to these decrements. Results indicate that these connexon-mediated interliposomal channels are functional and are inhibited by the addition of alpha-glycyrrhetinic acid and by flufenamic acid, two gap junction communication inhibitors. Future use of this model system has the potential to improve our understanding of the permeability and modulation of junctional channels in its native intercellular assembly.

  9. Chaos synchronization in gap-junction-coupled neurons

    NASA Astrophysics Data System (ADS)

    Yoshioka, Masahiko

    2005-06-01

    Depending on temperature, the modified Hodgkin-Huxley (MHH) equations exhibit a variety of dynamical behaviors, including intrinsic chaotic firing. We analyze synchronization in a large ensemble of MHH neurons that are interconnected with gap junctions. By evaluating tangential Lyapunov exponents we clarify whether the synchronous state of neurons is chaotic or periodic. Then, we evaluate transversal Lyapunov exponents to elucidate if this synchronous state is stable against infinitesimal perturbations. Our analysis elucidates that with weak gap junctions, the stability of the synchronization of MHH neurons shows rather complicated changes with temperature. We, however, find that with strong gap junctions, the synchronous state is stable over the wide range of temperature irrespective of whether synchronous state is chaotic or periodic. It turns out that strong gap junctions realize the robust synchronization mechanism, which well explains synchronization in interneurons in the real nervous system.

  10. Gap junctions/hemichannels modulate interkinetic nuclear migration in the forebrain precursors

    PubMed Central

    Liu, Xiuxin; Hashimoto-Torii, Kazue; Torii, Masaaki; Ding, Chen; Rakic, Pasko

    2010-01-01

    During mitotic division in the telencephalic proliferative ventricular zone (VZ), the nuclei of the neural precursors move basally away from the ventricular surface for DNA synthesis, and apically return to the surface for mitotic division; a process known as interkinetic migration or “to-and-fro” nuclear translocation. The cell, which remains attached to the ventricular surface, either continues cycling, or exits the cycle and migrates to the subventricular zone (SVZ) or the developing cortical plate. While gap junctions/hemichannels are known to modulate DNA synthesis via Ca2+ waves, the role of Ca+ oscillations and the mechanism of nuclear translocation in the VZ precursors are unclear. Here we provide evidence that during apical nuclear migration, VZ precursors display dynamic spontaneous Ca2+ transients, which depend on functional gap junctions/hemichannels via ATP release and Ca2+ mobilizing messenger diffusion. Furthermore, we found that blocking gap junctions/hemichannels or shRNA mediated knockdown of connexin 43 (Cx43) retards the apically directed interkinetic nuclear migration accompanied with changes in the nuclear length/width ratio. In addition, we demonstrated that blocking functional gap junctions/hemichannels induces phosphorylation of small GTPase cdc42 in the VZ precursors. The basal phase of interkinetic migration is much slower and appears to be mediated passively by mechanical forces after cell division. Our findings indicate that functional interference with gap junctions/hemichannels during embryonic development may lead to abnormal corticogenesis and dysfunction of the cerebral cortex in adult organisms. PMID:20335455

  11. Activated microglia do not form functional gap junctions in vivo.

    PubMed

    Wasseff, Sameh K; Scherer, Steven S

    2014-04-15

    We investigated whether microglia form gap junctions with themselves, or with astrocytes, oligodendrocytes, or neurons in vivo in normal mouse brains, and in pathological conditions that induce microglial activation - brain injury and a model of Alzheimer's disease. Although microglia are in close physical proximity to glia and neurons, they do not form functional gap junctions under these pathological conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Anchored PKA as a gatekeeper for gap junctions.

    PubMed

    Pidoux, Guillaume; Taskén, Kjetil

    2015-01-01

    Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types.

  13. 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

  14. Fixed-gap tunnel junction for reading DNA nucleotides.

    PubMed

    Pang, Pei; Ashcroft, Brian Alan; Song, Weisi; Zhang, Peiming; Biswas, Sovan; Qing, Quan; Yang, Jialing; Nemanich, Robert J; Bai, Jingwei; Smith, Joshua T; Reuter, Kathleen; Balagurusamy, Venkat S K; Astier, Yann; Stolovitzky, Gustavo; Lindsay, Stuart

    2014-12-23

    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.

  15. Reduction of Gap Junctional Conductance by Microinjection of Antibodies against the 27-kDa Liver Gap Junction Polypeptide

    NASA Astrophysics Data System (ADS)

    Hertzberg, E. L.; Spray, D. C.; Bennett, M. V. L.

    1985-04-01

    Antibody raised against isolated rat liver gap junctions was microinjected into coupled cells in culture to assess its influence on gap junctional conductance. A rapid inhibition of fluorescent dye transfer and electrical coupling was produced in pairs of freshly dissociated adult rat hepatocytes and myocardial cells as well as in pairs of superior cervical ganglion neurons from neonatal rats cultured under conditions in which electrotonic synapses form. The antibodies have been shown by indirect immunofluorescence to bind to punctate regions of the plasma membrane in liver. By immunoreplica analysis of rat liver homogenates, plasma membranes, and isolated gap junctions resolved on NaDodSO4/polyacrylamide gels, binding was shown to be specific for the 27-kDa major polypeptide of gap junctions. This and similar antibodies should provide a tool for further investigation of the role of cell-cell communication mediated by gap junctions and indicate that immunologically similar polypeptides comprise gap junctions in adult mammalian cells derived from all three germ layers.

  16. Role of gap junctions in the propagation of the cardiac action potential.

    PubMed

    Rohr, Stephan

    2004-05-01

    Gap junctions play a pivotal role for the velocity and the safety of impulse propagation in cardiac tissue. Under physiologic conditions, the specific subcellular distribution of gap junctions together with the tight packaging of the rod-shaped cardiomyocytes underlies anisotropic conduction, which is continuous at the macroscopic scale. However, when breaking down the three-dimensional network of cells into linear single cell chains, gap junctions can be shown to limit axial current flow and to induce 'saltatory' conduction at unchanged overall conduction velocities. In two- and three-dimensional tissue, these discontinuities disappear due to lateral averaging of depolarizing current flow at the activation wavefront. During gap junctional uncoupling, discontinuities reappear and are accompanied by slowed and meandering conduction. Critical gap junctional uncoupling reduces conduction velocities to a much larger extent than does a reduction of excitability, which suggests that the safety for conduction is higher at any given conduction velocity for gap junctional uncoupling. In uniformly structured tissue, gap junctional uncoupling is accompanied by a parallel decrease in conduction velocity. However, this is not necessarily the case for non-uniform structures like tissue expansion where partial uncoupling paradoxically increases conduction velocity and has the capacity to remove unidirectional conduction blocks. Whereas the impact of gap junctions on impulse conduction is generally assessed from the point of view of cell coupling among cardiomyocytes, it is possible that other cell types within the myocardium might be coupled to cardiomyocytes as well. In this context, it has been shown that fibroblasts establish successful conduction between sheets of cardiomyocytes over distances as long as 300 microm. This might not only explain electrical synchronization of heart transplants but might be of importance for cardiac diseases involving fibrosis. Finally, the

  17. Pallidal gap junctions-triggers of synchrony in Parkinson's disease?

    PubMed

    Schwab, Bettina C; Heida, Tjitske; Zhao, Yan; van Gils, Stephan A; van Wezel, Richard J A

    2014-10-01

    Although increased synchrony of the neural activity in the basal ganglia may underlie the motor deficiencies exhibited in Parkinson's disease (PD), how this synchrony arises, propagates through the basal ganglia, and changes under dopamine replacement remains unknown. Gap junctions could play a major role in modifying this synchrony, because they show functional plasticity under the influence of dopamine and after neural injury. In this study, confocal imaging was used to detect connexin-36, the major neural gap junction protein, in postmortem tissues of PD patients and control subjects in the putamen, subthalamic nucleus (STN), and external and internal globus pallidus (GPe and GPi, respectively). Moreover, we quantified how gap junctions affect synchrony in an existing computational model of the basal ganglia. We detected connexin-36 in the human putamen, GPe, and GPi, but not in the STN. Furthermore, we found that the number of connexin-36 spots in PD tissues increased by 50% in the putamen, 43% in the GPe, and 109% in the GPi compared with controls. In the computational model, gap junctions in the GPe and GPi strongly influenced synchrony. The basal ganglia became especially susceptible to synchronize with input from the cortex when gap junctions were numerous and high in conductance. In conclusion, connexin-36 expression in the human GPe and GPi suggests that gap junctional coupling exists within these nuclei. In PD, neural injury and dopamine depletion could increase this coupling. Therefore, we propose that gap junctions act as a powerful modulator of synchrony in the basal ganglia. © 2014 International Parkinson and Movement Disorder Society.

  18. New cellular mechanisms of gap junction degradation and recycling.

    PubMed

    Carette, Diane; Gilleron, Jérôme; Denizot, Jean-Pierre; Grant, Kirsty; Pointis, Georges; Segretain, Dominique

    2015-07-01

    Connexins (Cxs), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore, alterations of Cx expression and degradation lead to the development of physiopathological disorders. Because of the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear. By using a combination of time-lapse fluorescence microscopy and high-resolution transmission electron microscopy, we evidenced new specific cellular events concerning gap junction degradation and recycling. Indeed, by time lapse video microscopy we demonstrated, for the first time to our knowledge, that an entire AGJ can be fully recycled back to the plasma membrane. Moreover, we dissected the degradative processes of gap junction by electron microscopy approaches. Interestingly, in addition to canonical autophagy and heterophagy pathways, previously described, we discovered that both pathways could sometimes intermingle. Strikingly, our results also highlighted a new lysosome-based autophagy pathway that could play a pivotal role in common autophagy degradation. The present investigation reveals that AGJ degradation is a more complex process that it was previously thought. First, a complete recycling of the gap junction plaque after its internalisation could occur. Second, the degradation of this peculiar double membrane structure is possible through autophagy, heterophagy, hetero-autophagy or by lysosomal-based autophagy. Altogether, this work underlines novel aspects of gap junction degradation that could be extended to other cell biology processes. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  19. The role of gap junctions in stretch-induced atrial fibrillation.

    PubMed

    Ueda, Norihiro; Yamamoto, Mitsuru; Honjo, Haruo; Kodama, Itsuo; Kamiya, Kaichiro

    2014-11-01

    The aim of this study was to investigate the role of gap junctions in atrial fibrillation (AF) by analysing the effects of a gap junction enhancer and blocker on AF vulnerability and electrophysiological properties of isolated hearts. The acute atrial stretch model of AF in the isolated rabbit heart was used. Sustained AF (SAF) was induced by a burst of high-frequency stimulation of the Bachmann's bundle. The effective refractory period (ERP) was measured, and the total conduction time (TCT) and the pattern of conduction of the anterior surface of the left atrium were monitored by using an optical mapping system. The effect of enhancing gap junction function by 100-1000 nM rotigaptide (ZP123) and block by 30 μM carbenoxolone on these parameters was measured. SAF inducibility was increased with an elevation of intra-atrial pressure. Enhanced gap junction conductance induced by treatment with 100-1000 nM rotigaptide reduced SAF inducibility, and the gap junction blocker carbenoxolone increased SAF inducibility. In the absence of gap junction enhancer or blocker, normal conduction was observed at 0 cmH2O. When intra-atrial pressure was raised to 12 cmH2O, the conduction pattern was changed to a heterogeneous zig-zag pattern and TCT was prolonged. Conduction pattern was not affected by either agent. Rotigaptide shortened TCT, whereas carbenoxolone prolonged TCT. ERP was significantly shortened with an increase in intra-atrial pressure, but ERP was unaffected by either agent. Gap junction modulators changed AF inducibility through their effects on atrial conduction, not by altering ERP. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Cardiology.

  20. Dynamic gap junctional communication: a delimiting model for tissue responses.

    PubMed Central

    Christ, G J; Brink, P R; Ramanan, S V

    1994-01-01

    Gap junctions are aqueous intercellular channels formed by a diverse class of membrane-spanning proteins, known as connexins. These aqueous pores provide partial cytoplasmic continuity between cells in most tissues, and are freely permeable to a host of physiologically relevant second messenger molecules/ionic species (e.g., Ca2+, IP3, cAMP, cGMP). Despite the fact that these second messenger molecules/ionic species have been shown to alter junctional patency, there is no clear basis for understanding how dynamic and transient changes in the intracellular concentration of second messenger molecules might modulate the extent of intercellular communication among coupled cells. Thus, we have modified the tissue monolayer model of Ramanan and Brink (1990) to account for both the up-regulatory and down-regulatory effects on junctions by second messenger molecules that diffuse through gap junctions. We have chosen the vascular wall as our morphological correlate because of its anisotropy and large investment of gap junctions. The model allows us to illustrate the putative behavior of gap junctions under a variety of physiologically relevant conditions. The modeling studies demonstrated that transient alterations in intracellular second messenger concentrations are capable of producing 50-125% changes in the number of cells recruited into a functional syncytial unit, after activation of a single cell. Moreover, the model conditions required to demonstrate such physiologically relevant changes in intercellular diffusion among coupled cells are commonly observed in intact tissues and cultured cells. Images FIGURE 2 PMID:7811948

  1. Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions

    PubMed Central

    Thuringer, Dominique; Boucher, Jonathan; Jego, Gaetan; Pernet, Nicolas; Cronier, Laurent; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-01-01

    Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types. PMID:27661112

  2. Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions.

    PubMed

    Thuringer, Dominique; Boucher, Jonathan; Jego, Gaetan; Pernet, Nicolas; Cronier, Laurent; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-11-08

    Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types.

  3. Screening of gap junction antagonists on dye coupling in the rabbit retina

    PubMed Central

    PAN, FENG; MILLS, STEPHEN L.; MASSEY, STEPHEN C.

    2008-01-01

    Many cell types in the retina are coupled via gap junctions and so there is a pressing need for a potent and reversible gap junction antagonist. We screened a series of potential gap junction antagonists by evaluating their effects on dye coupling in the network of A-type horizontal cells. We evaluated the following compounds: meclofenamic acid (MFA), mefloquine, 2-aminoethyldiphenyl borate (2-APB), 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid (18-β-GA), retinoic acid, flufenamic acid, niflumic acid, and carbenoxolone. The efficacy of each drug was determined by measuring the diffusion coefficient for Neurobiotin (Mills & Massey, 1998). MFA, 18-β-GA, 2-APB and mefloquine were the most effective antagonists, completely eliminating A-type horizontal cell coupling at a concentration of 200 μM. Niflumic acid, flufenamic acid, and carbenoxolone were less potent. Additionally, carbenoxolone was difficult to wash out and also may be harmful, as the retina became opaque and swollen. MFA, 18-β-GA, 2-APB and mefloquine also blocked coupling in B-type horizontal cells and AII amacrine cells. Because these cell types express different connexins, this suggests that the antagonists were relatively non-selective across several different types of gap junction. It should be emphasized that MFA was water-soluble and its effects on dye coupling were easily reversible. In contrast, the other gap junction antagonists, except carbenoxolone, required DMSO to make stock solutions and were difficult to wash out of the preparation at the doses required to block coupling in A-type HCs. The combination of potency, water solubility and reversibility suggest that MFA may be a useful compound to manipulate gap junction coupling. PMID:17711600

  4. Gap Junctions in Developing Thalamic and Neocortical Neuronal Networks

    PubMed Central

    Niculescu, Dragos; Lohmann, Christian

    2014-01-01

    The presence of direct, cytoplasmatic, communication between neurons in the brain of vertebrates has been demonstrated a long time ago. These gap junctions have been characterized in many brain areas in terms of subunit composition, biophysical properties, neuronal connectivity patterns, and developmental regulation. Although interesting findings emerged, showing that different subunits are specifically regulated during development, or that excitatory and inhibitory neuronal networks exhibit various electrical connectivity patterns, gap junctions did not receive much further interest. Originally, it was believed that gap junctions represent simple passageways for electrical and biochemical coordination early in development. Today, we know that gap junction connectivity is tightly regulated, following independent developmental patterns for excitatory and inhibitory networks. Electrical connections are important for many specific functions of neurons, and are, for example, required for the development of neuronal stimulus tuning in the visual system. Here, we integrate the available data on neuronal connectivity and gap junction properties, as well as the most recent findings concerning the functional implications of electrical connections in the developing thalamus and neocortex. PMID:23843439

  5. Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

    PubMed

    Szoboszlay, Miklos; Lőrincz, Andrea; Lanore, Frederic; Vervaeke, Koen; Silver, R Angus; Nusser, Zoltan

    2016-06-01

    The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Gap junctions in developing thalamic and neocortical neuronal networks.

    PubMed

    Niculescu, Dragos; Lohmann, Christian

    2014-12-01

    The presence of direct, cytoplasmatic, communication between neurons in the brain of vertebrates has been demonstrated a long time ago. These gap junctions have been characterized in many brain areas in terms of subunit composition, biophysical properties, neuronal connectivity patterns, and developmental regulation. Although interesting findings emerged, showing that different subunits are specifically regulated during development, or that excitatory and inhibitory neuronal networks exhibit various electrical connectivity patterns, gap junctions did not receive much further interest. Originally, it was believed that gap junctions represent simple passageways for electrical and biochemical coordination early in development. Today, we know that gap junction connectivity is tightly regulated, following independent developmental patterns for excitatory and inhibitory networks. Electrical connections are important for many specific functions of neurons, and are, for example, required for the development of neuronal stimulus tuning in the visual system. Here, we integrate the available data on neuronal connectivity and gap junction properties, as well as the most recent findings concerning the functional implications of electrical connections in the developing thalamus and neocortex.

  7. Gap Junctions in the Control of Vascular Function

    PubMed Central

    Duling, Brian R.

    2009-01-01

    Abstract Direct intercellular communication via gap junctions is critical in the control and coordination of vascular function. In the cardiovascular system, gap junctions are made up of one or more of four connexin proteins: Cx37, Cx40, Cx43, and Cx45. The expression of more than one gap-junction protein in the vasculature is not redundant. Rather, vascular connexins work in concert, first during the development of the cardiovascular system, and then in integrating smooth muscle and endothelial cell function, and in coordinating cell function along the length of the vessel wall. In addition, connexin-based channels have emerged as an important signaling pathway in the astrocyte-mediated neurovascular coupling. Direct electrical communication between endothelial cells and vascular smooth muscle cells via gap junctions is thought to play a relevant role in the control of vasomotor tone, providing the signaling pathway known as endothelium-derived hyperpolarizing factor (EDHF). Consistent with the importance of gap junctions in the regulation of vasomotor tone and arterial blood pressure, the expression of connexins is altered in diseases associated with vascular complications. In this review, we discuss the participation of connexin-based channels in the control of vascular function in physiologic and pathologic conditions, with a special emphasis on hypertension and diabetes. Antioxid. Redox Signal. 11, 251–266. PMID:18831678

  8. 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

  9. Lighting up gap junction channels in a flash.

    PubMed

    Evans, W Howard; Martin, Patricia E M

    2002-10-01

    Gap junction intercellular communication channels permit the exchange of small regulatory molecules and ions between neighbouring cells and coordinate cellular activity in diverse tissue and organ systems. These channels have short half-lives and complex assembly and degradation pathways. Much of the recent work elucidating gap junction biogenesis has featured the use of connexins (Cx), the constituent proteins of gap junctions, tagged with reporter proteins such as Green Fluorescent Protein (GFP) and has illuminated the dynamics of channel assembly in live cells by high-resolution time-lapse microscopy. With some studies, however, there are potential short-comings associated with the GFP chimeric protein technologies. A recent report by Gaietta et al., has highlighted the use of recombinant proteins with tetracysteine tags attached to the carboxyl terminus of Cx43, which differentially labels 'old' and 'new' connexins thus opening up new avenues for studying temporal and spatial localisation of proteins and in situ trafficking events. Copyright 2002 Wiley Periodicals, Inc.

  10. Dynamic switching of neural codes in networks with gap junctions.

    PubMed

    Katori, Yuichi; Masuda, Naoki; Aihara, Kazuyuki

    2006-12-01

    Population rate coding and temporal coding are common neural codes. Recent studies suggest that these two codes may be alternatively used in one neural system. Based on the fact that there are massive gap junctions in the brain, we explore how this switching behavior may be related to neural codes in networks of neurons connected by gap junctions. First, we show that under time-varying inputs, such neural networks show switching between synchronous and asynchronous states. Then, we quantify network dynamics by three mutual information measures to show that population rate coding carries more information in asynchronous states and temporal coding does so in synchronous states.

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

    PubMed Central

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

    2001-01-01

    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. PMID:11720990

  12. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques

    PubMed Central

    1991-01-01

    We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43- NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form. PMID:1659577

  13. Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

    PubMed

    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

    2012-05-22

    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 examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. 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 before 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 connexin37 hemichannels and gap junctions in platelet thrombus function. Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

  14. [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.

  15. Dynamically Active Compartments Coupled by a Stochastically Gated Gap Junction

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Lawley, Sean D.

    2017-03-01

    We analyze a one-dimensional PDE-ODE system representing the diffusion of signaling molecules between two cells coupled by a stochastically gated gap junction. We assume that signaling molecules diffuse within the cytoplasm of each cell and then either bind to some active region of the cell's membrane (treated as a well-mixed compartment) or pass through the gap junction to the interior of the other cell. We treat the gap junction as a randomly fluctuating gate that switches between an open and a closed state according to a two-state Markov process. This means that the resulting PDE-ODE is stochastic due to the presence of a randomly switching boundary in the interior of the domain. It is assumed that each membrane compartment acts as a conditional oscillator, that is, it sits below a supercritical Hopf bifurcation. In the ungated case (gap junction always open), the system supports diffusion-induced oscillations, in which the concentration of signaling molecules within the two compartments is either in-phase or anti-phase. The presence of a reflection symmetry (for identical cells) means that the stochastic gate only affects the existence of anti-phase oscillations. In particular, there exist parameter choices where the gated system supports oscillations, but the ungated system does not, and vice versa. The existence of oscillations is investigated by solving a spectral problem obtained by averaging over realizations of the stochastic gate.

  16. TLR2 Regulates Gap Junction Intercellular Communication in Airway Cells

    PubMed Central

    Martin, Francis J.; Prince, Alice S.

    2009-01-01

    The innate immune response to inhaled bacteria, such as the opportunist Pseudomonas aeruginosa, is initiated by TLR2 displayed on the apical surface of airway epithelial cells. Activation of TLR2 is accompanied by an immediate Ca2+ flux that is both necessary and sufficient to stimulate NF-κB and MAPK proinflammatory signaling to recruit and activate polymorphonuclear leukocytes in the airway. In human airway cells gap junction channels were found to provide a regulated conduit for the movement of Ca2+ from cell to cell. In response to TLR2 stimulation, by either lipid agonists or P. aeruginosa, gap junctions functioned to transiently amplify proinflammatory signaling by communicating Ca2+ fluxes from stimulated to adjacent, non-stimulated cells thus increasing epithelial CXCL8 production. P. aeruginosa stimulation also induced tyrosine phosphorylation of Connexin 43 and association with c-Src, events linked to the closure of these channels. By 4 hours post bacterial stimulation, gap junction communication was decreased indicating an autoregulatory control of the connexins. Thus, gap junction channels comprised of Connexin 43 and other connexins in airway cells provide a mechanism to coordinate and regulate the epithelial immune response even in the absence of signals from the immune system. PMID:18354224

  17. The psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.

    PubMed

    Liu, Xinhe; Petit, Jean-Marie; Ezan, Pascal; Gyger, Joël; Magistretti, Pierre; Giaume, Christian

    2013-12-01

    Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

  18. Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells.

    PubMed

    Takeda, Yukari; Ward, Sean M; Sanders, Kenton M; Koh, Sang Don

    2005-04-01

    In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.

  19. Requirement of gap junctional intercellular communication for human villous trophoblast differentiation.

    PubMed

    Cronier, Laurent; Frendo, Jean-Louis; Defamie, Norah; Pidoux, Guillaume; Bertin, Gladys; Guibourdenche, Jean; Pointis, Georges; Malassine, Andre

    2003-11-01

    During pregnancy, the villous trophoblast develops from the fusion of cytotrophoblastic cells (CT) into a syncytiotrophoblast (ST), supporting the main physiological functions of the human placenta. Connexin43 (Cx43) is demonstrated in situ and in vitro in the villous trophoblast between CT and between CT and ST. Moreover, the presence of a gap junctional intercellular communication (GJIC) during in vitro trophoblast differentiation was previously demonstrated. Because the exchange of molecules through gap junctions is considered to play a major role in the control of cell and tissue differentiation, we studied the effects of a gap junctional uncoupler, heptanol, on morphological and functional trophoblast differentiation and on GJIC measured by the fluorescence recovery after photobleaching method. We found that when the GJIC was interrupted, CT still aggregated but fused poorly. This morphological effect was associated with a significant decrease of trophoblastic-specific gene expression (beta human chorionic gonadotropin and human chorionic somatomammotropin). This blocking action was reversible as demonstrated by recovery of GJIC and trophoblast differentiation process after heptanol removal. Moreover, the inhibition of the trophoblast differentiation did not affect Cx43 transcript expression and Cx43 protein expression. These data suggest that the molecular exchanges through gap junctions preceding cellular fusion are essential for trophoblast differentiation generating the multifunctional syncytiotrophoblast.

  20. Aquaporin 0 enhances gap junction coupling via its cell adhesion function and interaction with connexin 50

    PubMed Central

    Liu, Jialu; Xu, Ji; Gu, Sumin; Nicholson, Bruce J.; Jiang, Jean X.

    2011-01-01

    Both connexin 50 (Cx50) and aquaporin 0 (AQP0) have important roles in lens development and homeostasis, and their mutations are associated with human congenital cataracts. We have previously shown that Cx50 directly interacts with AQP0. Here, we demonstrate the importance of the Cx50 intracellular loop (IL) domain in mediating the interaction with AQP0 in the lens in vivo. AQP0 significantly increased (~20–30%) the intercellular coupling and conductance of Cx50 gap junctions. However, this increase was not observed when the IL domain was replaced with those from other lens connexins. The Cx50–AQP0 interaction had no effect on Cx50 hemichannel function. A fusion protein containing three extracellular loop domains of AQP0 efficiently blocked the cell-to-cell adhesion of AQP0 and attenuated the stimulatory effect of AQP0 on Cx50 gap junction conductance. These data suggest that the specific interaction between Cx50 and AQP0 enhances the coupling of Cx50 gap junctions, but not hemichannels, through the cell adhesion function of AQP0. This result establishes a physiological role of AQP0 in the functional regulation of gap junction channels. PMID:21172802

  1. Gap junctions and tissue business: problems and strategies for developing specific functional reagents.

    PubMed

    Goodenough, D A; Musil, L S

    1993-01-01

    The complex and overlapping tissue distribution of different members of the gap junctional connexin protein family is reviewed. Intermixing of different connexins in the building of intercellular channels and translational and posttranslational regulation of gap junctional channels add additional challenges to the interpretation of the possible functions played by gap junction-mediated intercellular communication in tissue business.

  2. Length and energy gap dependences of thermoelectricity in nanostructured junctions.

    PubMed

    Asai, Yoshihiro

    2013-04-17

    The possibilities of an enhanced thermoelectric figure of merit value, ZT, in a nanostructured junction are examined for a wide range of parameter values in a theoretical model. Our research shows that the figure of merit can take a very large maximum, which depends both on the length and the energy gap values. The maximum of ZT is achieved when the Fermi level of the electrodes is aligned to the edge of the electronic transmission function of the junction, where both the conductance and the Seebeck constant are significantly enhanced. On the basis of our results, we conclude that nanowires and molecular junctions form a special class of systems where a large ZT can be expected in some cases.

  3. TLR-Activated Gap Junction Channels Protect Mice against Bacterial Infection through Extracellular UDP Release.

    PubMed

    Qin, Juliang; Zhang, Guangxu; Zhang, Xiaoyu; Tan, Binghe; Lv, Zhangsheng; Liu, Mingyao; Ren, Hua; Qian, Min; Du, Bing

    2016-02-15

    Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  4. Gap junctions: their importance for the dynamics of neural circuits.

    PubMed

    Rela, Lorena; Szczupak, Lidia

    2004-12-01

    Electrical coupling through gap junctions constitutes a mode of signal transmission between neurons (electrical synaptic transmission). Originally discovered in invertebrates and in lower vertebrates, electrical synapses have recently been reported in immature and adult mammalian nervous systems. This has renewed the interest in understanding the role of electrical synapses in neural circuit function and signal processing. The present review focuses on the role of gap junctions in shaping the dynamics of neural networks by forming electrical synapses between neurons. Electrical synapses have been shown to be important elements in coincidence detection mechanisms and they can produce complex input-output functions when arranged in combination with chemical synapses. We postulate that these synapses may also be important in redefining neuronal compartments, associating anatomically distinct cellular structures into functional units. The original view of electrical synapses as static connecting elements in neural circuits has been revised and a considerable amount of evidence suggests that electrical synapses substantially affect the dynamics of neural circuits.

  5. Gap Junctional Regulation of Signal Transduction in Bone Cells

    PubMed Central

    Buo, Atum M.; Stains, Joseph P.

    2014-01-01

    The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions. PMID:24486014

  6. Role of gap junctions modulating hepatic vascular tone in cirrhosis.

    PubMed

    Hernández-Guerra, Manuel; González-Méndez, Yanira; de Ganzo, Zaida A; Salido, Eduardo; García-Pagán, Juan C; Abrante, Beatriz; Malagón, Antonio M; Bosch, Jaime; Quintero, Enrique

    2014-07-01

    Gap junctions are formed by connexins (Cx), a family of proteins that couple endothelial and smooth muscle cells in systemic vessels. In this context, Cx allow the transmission of signals modulating vascular tone. Recently, vascular Cx have been observed in liver cells implicated in liver blood flow regulation. Here, we investigated the role of Cx in the regulation of intrahepatic vascular tone in cirrhosis. Livers of Sprague-Dawley control and cirrhotic (common bile duct ligation-CBDL and CCl4 ) rats were perfused, and concentration-effect curves in response to acetylcholine (ACh) precontracted with methoxamine were obtained in the presence of the specific Cx inhibitor 18-alpha-glycyrrhetinic acid or vehicle. Cx expression was assessed by immunofluorescence, western blot and reverse-transcription polymerase chain reaction in liver tissue, hepatic stellate cells, sinusoidal endothelial cells and hepatocytes isolated from control and cirrhotic rat livers. Cx protein expression was also determined in cirrhotic human tissue. Gap junction blockade markedly attenuated relaxation of hepatic vasculature in response to ACh in control (maximal relaxation, -55 ± 10.5% vs. -95.3 ± 10% with vehicle; P < 0.01) and CBDL rats (50.9 ± 18.5% vs. -18.7 ± 5.5% with vehicle; P = 0.01). Livers from CBDL rats and patients with cirrhosis exhibited Cx overexpression. By contrast, CCl4 -cirrhotic rats did not show attenuated relaxation of hepatic vasculature after blockade and Cx expression was significantly lower than in controls. Gap junctions may contribute to modulating portal pressure and intrahepatic vascular relaxation. Liver gap junctions may represent a new therapeutic target in cirrhotic portal hypertension. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Gap Junction Intercellular Communication in Bone Marrow Failure

    DTIC Science & Technology

    2012-10-01

    osteogenesis, osteoblast function, or mesenchymal-derived hematopoietic support. This project postulates that the loss of gap junction ( GJ )-mediated...intercellular communication (IC) in the osteogenic HM is one of the mechanisms involved in dysfunctional mesenchymal hematopoietic support. GJ are cell...bone (30-39). GJ are formed by hexamers (hemichannels) of a family of proteins called connexins, with cell specific expression and functions(40, 41

  8. Myosin VI facilitates connexin 43 gap junction accretion

    PubMed Central

    Waxse, Bennett J.

    2017-01-01

    ABSTRACT 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. PMID:28096472

  9. Gap junctions synchronize the firing of inhibitory interneurons in guinea pig hippocampus.

    PubMed

    Yang, Q; Michelson, H B

    2001-07-13

    The convulsant 4-aminopyridine (4AP) facilitates the synchronous firing of interneurons in the hippocampus, eliciting giant inhibitory postsynaptic potentials (IPSPs) in CA3 pyramidal cells. We used the gap junction blocker carbenoxolone to investigate the role of electrotonic coupling in both the initiation and the maintenance of 4AP-facilitated inhibitory circuit oscillations. Carbenoxolone abolished all synchronized IPSPs in CA3 cells elicited by 4AP in the presence of ionotropic glutamate receptor blockers. Carbenoxolone also blocked the isolated synchronized GABA(B) IPSPs generated in CA3 cells by a subpopulation of interneurons. These data confirm that: (1) the interneurons producing GABA(B) responses in CA3 cells are electrotonically coupled, and (2) gap junctions among interneurons are essential for initiating synchronized interneuron oscillatory firing in 4AP.

  10. 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

  11. 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.

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

    PubMed Central

    Willebrords, Joost; Vinken, Mathieu

    2015-01-01

    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. PMID:26420514

  13. Regulation of gap junction coupling in bovine ciliary epithelium

    PubMed Central

    Wang, Zhao; Do, Chi Wai; Valiunas, Virginijus; Leung, Chi Ting; Cheng, Angela K. W.; Clark, Abbott F.; Wax, Martin B.; Chatterton, Jon E.

    2010-01-01

    Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 (n = 15) to 0.17 ± 0.05 (n = 20) after 10 min. Junctional currents were lowered by ∼50% (n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication. PMID:20089928

  14. 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...

  15. 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...

  16. Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

    PubMed Central

    Moreno, A P; de Carvalho, A C; Verselis, V; Eghbali, B; Spray, D C

    1991-01-01

    We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134). PMID:1648416

  17. Gap junction structures. V. Structural chemistry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility.

    PubMed

    Makowski, L; Caspar, D L; Phillips, W C; Goodenough, D A

    1984-04-15

    X-ray diffraction patterns have been recorded from partially oriented specimens of gap junctions isolated from mouse liver and suspended in sucrose solutions of different concentration and thus of different electron density. Analysis of these diffraction patterns has shown that sucrose is excluded from the 6-fold rotation axis of the junction lattice for a length of about 100 A. This indicates that the aqueous channel of the junctions is in the closed, high resistance state in these preparations. Mapping of the sucrose-accessible space in the junction indicates that the cross-sectional area of the channel entrance on the cytoplasmic side of the membrane could be up to five times larger than the area of the transmembrane channel. Sucrose does not penetrate more than 20 A into the membrane along the channel. Apparently the aqueous channel, 8 to 10 A in radius for most of its length, is narrowed or blocked by a small feature about 50 A from the center of the gap. Very close interactions exist between the gap junction protein and the lipid polar head groups on the cytoplasmic surface of the membrane. In this region, the protein intercalates between the polar head groups. These results suggest that the gap junction protein may have a functional two-domain structure. One domain, with a molecular weight of about 15,000, spans one bilayer and half of the gap and is contained largely within a radius of 25 A from the 6-fold axis. The second domain is smaller and occupies the cytoplasmic surface of the gap junction membrane. Trypsin digestion removes about 4000 Mr from the cytoplasmic surface domain of the junction protein. Most of the material susceptible to trypsin digestion is located more than 28 A from the 6-fold axis.

  18. Reversible interruption of gap junctional communication by testosterone propionate in cultured Sertoli cells and cardiac myocytes.

    PubMed

    Pluciennik, F; Verrecchia, F; Bastide, B; Hervé, J C; Joffre, M; Délèze, J

    1996-02-01

    A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 microM and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state.

  19. Expression and role of connexin-based gap junctions in pulmonary inflammatory diseases.

    PubMed

    Freund-Michel, Véronique; Muller, Bernard; Marthan, Roger; Savineau, Jean-Pierre; Guibert, Christelle

    2016-08-01

    Connexins are transmembrane proteins that can generate intercellular communication channels known as gap junctions. They contribute to the direct movement of ions and larger cytoplasmic solutes between various cell types. In the lung, connexins participate in a variety of physiological functions, such as tissue homeostasis and host defence. In addition, emerging evidence supports a role for connexins in various pulmonary inflammatory diseases, such as asthma, pulmonary hypertension, acute lung injury, lung fibrosis or cystic fibrosis. In these diseases, the altered expression of connexins leads to disruption of normal intercellular communication pathways, thus contributing to various pathophysiological aspects, such as inflammation or tissue altered reactivity and remodeling. The present review describes connexin structure and organization in gap junctions. It focuses on connexins in the lung, including pulmonary bronchial and arterial beds, by looking at their expression, regulation and physiological functions. This work also addresses the issue of connexin expression alteration in various pulmonary inflammatory diseases and describes how targeting connexin-based gap junctions with pharmacological tools, synthetic blocking peptides or genetic approaches, may open new therapeutic perspectives in the treatment of these diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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.

  1. Switch in Gap Junction Protein Expression is Associated with Selective Changes in Junctional Permeability During Keratinocyte Differentiation

    NASA Astrophysics Data System (ADS)

    Brissette, Janice L.; Kumar, Nalin M.; Gilula, Norton B.; Hall, James E.; Dotto, G. Paolo

    1994-07-01

    Gap junctional communication provides a mechanism for regulating multicellular activities by allowing the exchange of small diffusible molecules between neighboring cells. The diversity of gap junction proteins may exist to form channels that have different permeability properties. We report here that induction of terminal differentiation in mouse primary keratinocytes by calcium results in a specific switch in gap junction protein expression. Expression of α_1 (connexin 43) and β_2 (connexin 26) gap junction proteins is down-modulated, whereas that of β_3 (connexin 31) and β_4 (connexin 31.1) proteins is induced. Although both proliferating and differentiating keratinocytes are electrically coupled, there are significant changes in the permeability properties of the junctions to small molecules. In parallel with the changes in gap junction protein expression during differentiation, the intercellular transfer of the small dyes neurobiotin, carboxyfluorescein, and Lucifer yellow is significantly reduced, whereas that of small metabolites, such as nucleotides and amino acids, proceeds unimpeded. Thus, a switch in gap junction protein expression in differentiating keratinocytes is accompanied by selective changes in junctional permeability that may play an important role in the coordinate control of the differentiation process.

  2. Conduction abnormalities and ventricular arrhythmogenesis: The roles of sodium channels and gap junctions.

    PubMed

    Tse, Gary; Yeo, Jie Ming

    2015-12-07

    Ventricular arrhythmias arise from disruptions in the normal orderly sequence of electrical activation and recovery of the heart. They can be categorized into disorders affecting predominantly cellular depolarization or repolarization, or those involving action potential (AP) conduction. This article briefly discusses the factors causing conduction abnormalities in the form of unidirectional conduction block and reduced conduction velocity (CV). It then examines the roles that sodium channels and gap junctions play in AP conduction. Finally, it synthesizes experimental results to illustrate molecular mechanisms of how abnormalities in these proteins contribute to such conduction abnormalities and hence ventricular arrhythmogenesis, in acquired pathologies such as acute ischaemia and heart failure, as well as inherited arrhythmic syndromes.

  3. 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

  4. Role of Gap Junctions and Hemichannels in Parasitic Infections

    PubMed Central

    Subiabre, Mario; Figueroa, Felipe; Schalper, Kurt Alex; Osorio, Luis; González, Jorge; Sáez, Juan Carlos

    2013-01-01

    In vertebrates, connexins (Cxs) and pannexins (Panxs) are proteins that form gap junction channels and/or hemichannels located at cell-cell interfaces and cell surface, respectively. Similar channel types are formed by innexins in invertebrate cells. These channels serve as pathways for cellular communication that coordinate diverse physiologic processes. However, it is known that many acquired and inherited diseases deregulate Cx and/or Panx channels, condition that frequently worsens the pathological state of vertebrates. Recent evidences suggest that Cx and/or Panx hemichannels play a relevant role in bacterial and viral infections. Nonetheless, little is known about the role of Cx- and Panx-based channels in parasitic infections of vertebrates. In this review, available data on changes in Cx and gap junction channel changes induced by parasitic infections are summarized. Additionally, we describe recent findings that suggest possible roles of hemichannels in parasitic infections. Finally, the possibility of new therapeutic designs based on hemichannel blokers is presented. PMID:24236292

  5. Gap-Junction Channels Dysfunction in Deafness and Hearing Loss

    PubMed Central

    Acuña, Rodrigo; Figueroa, Vania; Maripillan, Jaime; Nicholson, Bruce

    2009-01-01

    Abstract Gap-junction channels connect the cytoplasm of adjacent cells, allowing the diffusion of ions and small metabolites. They are formed at the appositional plasma membranes by a family of related proteins named connexins. Mutations in connexins 26, 31, 30, 32, and 43 have been associated with nonsyndromic or syndromic deafness. The majority of these mutations are inherited in an autosomal recessive manner, but a few of them have been associated with dominantly inherited hearing loss. Mutations in the connexin26 gene (GJB2) are the most common cause of genetic deafness. This review summarizes the most relevant and recent information about different mutations in connexin genes found in human patients, with emphasis on GJB2. The possible effects of the mutations on channel expression and function are discussed, in addition to their possible physiologic consequences for inner ear physiology. Finally, we propose that connexin channels (gap junctions and hemichannels) may be targets for age-related hearing loss induced by oxidative damage. Antioxid. Redox Signal. 11, 309–322. PMID:18837651

  6. Reduction in gap junction intercellular communication promotes glioma migration

    PubMed Central

    Aftab, Qurratulain; Sin, Wun-Chey; Naus, Christian C.

    2015-01-01

    Glioblastoma Multiforme (GBM), an aggressive form of adult brain tumor, is difficult to treat due to its invasive nature. One of the molecular changes observed in GBM is a decrease in the expression of the gap junction protein Connexin43 (Cx43); however, how a reduction in Cx43 expression contributes to glioma malignancy is still unclear. In this study we examine whether a decrease in Cx43 protein expression has a role in enhanced cell migration, a key feature associated with increased tumorigenicity. We used a 3D spheroid migration model that mimics the in vivo architecture of tumor cells to quantify migration changes. We found that down-regulation of Cx43 expression in the U118 human glioma cell line increased migration by reducing cell-ECM adhesion, and changed the migration pattern from collective to single cell. In addition gap junction intercellular communication (GJIC) played a more prominent role in mediating migration than the cytoplasmic interactions of the C-terminal tail. Live imaging revealed that reducing Cx43 expression enhanced relative migration by increasing the cell speed and affecting the direction of migration. Taken together our findings reveal an unexplored role of GJIC in facilitating collective migration. PMID:25926558

  7. Mefloquine gap junction blockade and risk of pregnancy loss.

    PubMed

    Nevin, Remington Lee

    2012-09-01

    Obstetric use of the antimalarial drug mefloquine has historically been discouraged during the first trimester and immediately before conception owing to concerns of potential fetal harm. With the rise of resistance to the antimalarial drug sulfadoxine-pyrimethamine (SP), mefloquine is now being considered as a replacement for SP for universal antenatal administration to women from malaria-endemic regions. Recent recommendations have also suggested that mefloquine may be used cautiously among pregnant travelers who cannot otherwise avoid visiting these areas. Mefloquine has been demonstrated to cause blockade of gap junction protein alpha 1 (GJA1) gap junction intercellular communication (GJIC), and recent evidence suggests that GJA1 GJIC is critical to successful embryonic implantation and early placental development. During routine use, mefloquine accumulates in organ and peripheral tissue, crosses the blood-placental barrier, and may plausibly accumulate in developing decidua and trophoblast at concentrations sufficient to interfere with GJA1 GJIC and, thus, cause deleterious effects on fetal outcomes. This conclusion is supported by epidemiological evidence that demonstrates use of the drug during early development is associated with an increased risk of miscarriage and stillbirth. Confirmatory studies are pending, but the available experimental and epidemiological evidence support renewed adherence, where feasible, to existing mefloquine package insert guidance that women avoid the drug during the periconceptional period.

  8. Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity

    NASA Technical Reports Server (NTRS)

    Claassen, D. E.; Spooner, B. S.

    1989-01-01

    Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

  9. Regulation of Cx43 gap junctions: the gatekeeper and the password.

    PubMed

    Hossain, M Z; Boynton, A L

    2000-10-17

    Gap junctions are regulatable pores that connect the cytoplasms of neighboring cells. Hossain and Boynton focus on connexin 43 gap junctions and their regulation by changing the phosphorylation status of the COOH-terminal domain of connexin 43 or by altering protein-protein interactions in this region. The COOH-terminal domain of connexin 43 appears to be a key player in regulating gap junctional communication (GJC) because many divergent signals in many different cell types modify this domain to inhibit GJC.

  10. Effects of microgravity on liposome-reconstituted cardiac gap junction channeling activity

    NASA Technical Reports Server (NTRS)

    Claassen, D. E.; Spooner, B. S.

    1989-01-01

    Effects of microgravity on cardiac gap junction channeling activity were investigated aboard NASA zero-gravity aircraft. Liposome-reconstituted gap junctions were assayed for channel function during free-fall, and the data were compared with channeling at 1 g. Control experiments tested for 0 g effects on the structural stability of liposomes, and on the enzyme-substrate signalling system of the assay. The results demonstrate that short periods of microgravity do not perturb reconstituted cardiac gap junction channeling activity.

  11. 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.

  12. Gap junctions are involved in the early generation of left-right asymmetry.

    PubMed

    Levin, M; Mercola, M

    1998-11-01

    Invariant left-right asymmetry of the visceral organs is a fundamental feature of vertebrate embryogenesis. While a cascade of asymmetrically expressed genes has been described, the embryonic mechanism that orients the left-right axis relative to the dorsoventral and anteroposterior axes (a prerequisite for asymmetric gene expression) is unknown. We propose that this process involves dorsoventral differences in cell-cell communication through gap junctions composed of connexin proteins. Global modulation of gap junctional states in Xenopus embryos by pharmacological agents specifically induced heterotaxia involving mirror-image reversals of heart, gut, and gall bladder. Greatest sensitivity was observed between st. 5 and st. 12, well before the onset of organogenesis. Moreover, heterotaxia was also induced following microinjection of dominant negative and wild-type connexin mRNAs to modify the endogenous dorsoventral difference in junctional communication. Heterotaxia was induced by either blocking gap junction communication (GJC) dorsally or by introducing communication ventrally (but not the reverse). Both connexin misexpression and exposure to GJC-modifying drugs altered expression of the normally left-sided gene XNR-1, demonstrating that GJC functions upstream of XNR-1 in the pathway that patterns left-right asymmetry. Finally, lineage analysis to follow the progeny of microinjected cells indicated that they generally do not contribute the asymmetric organs. Together with the early sensitivity window, this suggests that GJC functions as part of a fundamental, early aspect of left-right patterning. In addition, we show that a potential regulatory mutation in Connexin43 is sufficient to cause heterotaxia. Despite uncertainty about the prevalence of the serine364 to proline substitution reported in human patients with laterality defects, the mutant protein is both a mild hypomorph and a potent antimorph as determined by the effect of its expression on left

  13. 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.

  14. Gap junction structures. VIII. Membrane cross-sections.

    PubMed

    Sosinsky, G E; Jésior, J C; Caspar, D L; Goodenough, D A

    1988-05-01

    Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.

  15. The organization of adherens junctions and desmosomes at the cardiac intercalated disc is independent of gap junctions.

    PubMed

    Gutstein, David E; Liu, Fang-Yu; Meyers, Marian B; Choo, Andrew; Fishman, Glenn I

    2003-03-01

    Adherens junctions and desmosomes are responsible for mechanically coupling myocytes in the heart and are found closely apposed to gap junction plaques at the intercalated discs of cardiomyocytes. It is not known whether loss of cardiac gap junctions, such as described in cardiac disease states, may influence the expression patterns of other intercalated disc-associated proteins. We investigated whether the major cardiac gap junction protein connexin43 (Cx43) may be responsible for regulating adherens junctions, desmosomes and their associated catenins, in terms of abundance and localization at the intercalated discs of cardiomyocytes. In order to study the effect of loss of cardiac gap junctions on the intercalated disc-associated proteins, we used a combination of immunoblotting, immunofluorescence with confocal microscopy and electron microscopy to evaluate heart tissue from mice with cardiac-specific conditional knockout of Cx43. We found that the cardiac adherens junctions, desmosomes and their associated catenins, as well as vinculin and ZO-1, maintain their normal abundance, structural appearance and localization in the absence of Cx43. We conclude from these data that Cx43 is not required for the organization of the cell adhesion junctions and their associated catenins at the intercalated disc in the adult cardiac myocyte.

  16. Chlorpromazine reduces the intercellular communication via gap junctions in mammalian cells

    SciTech Connect

    Orellana, Juan A.; Palacios-Prado, Nicolas; Saez, Juan C. . E-mail: jsaez@bio.puc.cl

    2006-06-15

    In the work presented herein, we evaluated the effect of chlorpromazine (CPZ) on gap junctions expressed by two mammalian cell types; Gn-11 cells (cell line derived from mouse LHRH neurons) and rat cortical astrocytes maintained in culture. We also attempted to elucidate possible mechanisms of action of CPZ effects on gap junctions. CPZ, in concentrations comparable with doses used to treat human diseases, was found to reduce the intercellular communication via gap junctions as evaluated with measurements of dye coupling (Lucifer yellow). In both cell types, maximal inhibition of functional gap junctions was reached within about 1 h of treatment with CPZ, an recovery was almost complete at about 5 h after CPZ wash out. In both cell types, CPZ treatment increased the phosphorylation state of connexin43 (Cx43), a gap junction protein subunit. Moreover, CPZ reduced the reactivity of Cx43 (immunofluorescence) at cell interfaces and concomitantly increased its reactivity in intracellular vesicles, suggesting an increased retrieval from and/or reduced insertion into the plasma membrane. CPZ also caused cellular retraction reducing cell-cell contacts in a reversible manner. The reduction in contact area might destabilize existing gap junctions and abrogate formation of new ones. Moreover, the CPZ-induced reduction in gap junctional communication may depend on the connexins (Cxs) forming the junctions. If Cx43 were the only connexin expressed, MAPK-dependent phosphorylation of this connexin would induce closure of gap junction channels.

  17. Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells

    PubMed Central

    Thuringer, Dominique; Berthenet, Kevin; Cronier, Laurent; Jego, Gaetan; Solary, Eric; Garrido, Carmen

    2015-01-01

    High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth. PMID:25868858

  18. Gap junction-mediated electrical transmission: regulatory mechanisms and plasticity

    PubMed Central

    Pereda, Alberto E.; Curti, Sebastian; Hoge, Gregory; Cachope, Roger; Flores, Carmen E.; Rash, John E.

    2012-01-01

    The term synapse applies to cellular specializations that articulate the processing of information within neural circuits by providing a mechanism for the transfer of information between two different neurons. There are two main modalities of synaptic transmission: chemical and electrical. While most efforts have been dedicated to the understanding of the properties and modifiability of chemical transmission, less is still known regarding the plastic properties of electrical synapses, whose structural correlate is the gap junction. A wealth of data indicates that, rather than passive intercellular channels, electrical synapses are more dynamic and modifiable than was generally perceived. This article will discuss the factors determining the strength of electrical transmission and review current evidence demonstrating its dynamic properties. Like their chemical counterparts, electrical synapses can also be plastic and modifiable. PMID:22659675

  19. 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

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

    PubMed

    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.

  1. 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

  2. Gap junction proteins in the light-damaged albino rat

    PubMed Central

    Guo, Cindy X.; Tran, Henry; Green, Colin R.; Danesh-Meyer, Helen V.

    2014-01-01

    Purpose Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. Methods Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Results Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. Conclusions The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression. PMID:24883012

  3. Distribution of gap junctions and square array junctions in the mammalian lens.

    PubMed

    Costello, M J; McIntosh, T J; Robertson, J D

    1989-05-01

    The morphology of membrane specializations of the cortex and nucleus of bovine lenses has been analyzed for both isolated membrane fractions and intact tissue fragments. Fractions of fiber cell membranes isolated from the outer cortex and the inner nucleus of lenses have been compared using x-ray diffraction, electron microscopy, SDS polyacrylamide gels and Western blots. Each fraction has distinctive structural characteristics. In x-ray experiments, the cortical fraction gives no sharp equatorial reflections (from the plane of the membrane), whereas the nuclear fraction gives sharp equatorial reflections which index on a square lattice of 6.6 nm. In thin-section electron micrographs, the cortical fraction is composed primarily of closed vesicles and flat membrane sheets, some of which contain pentalamellar structures similar in appearance to the 16-18 nm thick gap junctions found in other tissues. The nuclear fraction contains mostly undulating membrane pairs which often show 11-14 nm pentalamellar profiles and occasionally thicker junctions. In freeze-fracture images the cortical membranes display irregular clusters of intramembrane particles which resemble gap junctions, whereas the nuclear membranes contain numerous large square arrays with a 6.6 nm repeat and few irregular clusters or individual intramembrane particles. Images of fragments of intact lenses used in the membrane isolations give similar results; in the cortex the area covered by gap junctions is over 50 times the area covered by square lattices, whereas nuclear fiber cell membranes contain large square arrays. Thus, cortical and nuclear fiber cell membranes have quite different morphologies. In particular, the size of the square arrays of protein increases as the fiber cells mature. SDS polyacrylamide gels from cortical and nuclear fractions are similar in that they both contain MP26 as the major band. However, Western blot analysis shows increasing quantities of lower molecular weight, 25 kD and

  4. Benzalkonium Chloride Suppresses Rabbit Corneal Endothelium Intercellular Gap Junction Communication

    PubMed Central

    Zhang, Zhenhao; Huang, Yue; Xie, Hui; Pan, Juxin; Liu, Fanfei; Li, Xuezhi; Chen, Wensheng; Hu, Jiaoyue; Liu, Zuguo

    2014-01-01

    Purpose Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects. Methods Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity. Results Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity. Conclusions The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations

  5. Effect of antipeptide antibodies directed against three domains of connexin43 on the gap junctional permeability of cultured heart cells.

    PubMed

    Bastide, B; Jarry-Guichard, T; Briand, J P; Délèze, J; Gros, D

    1996-04-01

    Cell-to-cell communication can be blocked by intracellular injections of antibodies raised against gap junction proteins, but the mechanism of channel obstruction is unknown. Binding to connexins could lead to a conformational change, interfere with regulatory domains or cause a steric hindrance. To address these questions, the effects on cell-to-cell communication of affinity purified polyclonal antibodies raised against peptides reproducing the intracellular sequences 5-17, 314-322 and 363-382 of rat connexin43 were investigated in cultured rat ventricular cells. The antibodies against sequence 363-382 were characterized by immunoblotting and immunocytochemistry. Characterization of antibodies 5-17 and 314-322 has been previously reported. In a first series of experiments, the effect on gap junctional communication was assessed by injecting a junction-permeant fluorescent dye into cells adjacent to one cell previously microinjected with antibodies. In a second series, junctional permeability was quantitatively determined on records of fluorescence recovery after the photobleaching of 6-carboxyfluorescein-loaded cells. Antibodies 5-17 marked a 43 kDa band on immunoblots, but did not immunolabel gap junctions and had no functional effect. Antibodies 314-322 recognized the 43 kDa protein and labeled the intercalated disks, but failed to interfere with junctional permeability. Antibodies to the nearby sequence 363-382, for which all immunospecific tests had been positive, caused a delayed diffusional uncoupling in 50% of the microinjected cells. It is suggested that the blocking of junctional communication by antibodies results from interference with a regulatory domain of the connexin.

  6. Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions

    PubMed Central

    1988-01-01

    Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4- 6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7- 6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7- 6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques. PMID:3372591

  7. 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

  8. 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...

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

    PubMed

    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.

  10. 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...

  11. Gap junctions facilitate propagation of synchronous firing in the cortical neural population: a numerical simulation study.

    PubMed

    Shinozaki, Takashi; Naruse, Yasushi; Câteau, Hideyuki

    2013-10-01

    This study investigates the effect of gap junctions on firing propagation in a feedforward neural network by a numerical simulation with biologically plausible parameters. Gap junctions are electrical couplings between two cells connected by a binding protein, connexin. Recent electrophysiological studies have reported that a large number of inhibitory neurons in the mammalian cortex are mutually connected by gap junctions, and synchronization of gap junctions, spread over several hundred microns, suggests that these have a strong effect on the dynamics of the cortical network. However, the effect of gap junctions on firing propagation in cortical circuits has not been examined systematically. In this study, we perform numerical simulations using biologically plausible parameters to clarify this effect on population firing in a feedforward neural network. The results suggest that gap junctions switch the temporally uniform firing in a layer to temporally clustered firing in subsequent layers, resulting in an enhancement in the propagation of population firing in the feedforward network. Because gap junctions are often modulated in physiological conditions, we speculate that gap junctions could be related to a gating function of population firing in the brain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. 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.

  13. 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

  14. 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...

  15. Differential regulation of the levels of three gap junction mRNAs in Xenopus embryos

    PubMed Central

    1990-01-01

    Xenopus mRNAs that potentially encode gap junction proteins in the oocyte and early embryo have been identified by low-stringency screening of cDNA libraries with cloned mammalian gap junction cDNAs. The levels of these mRNAs show strikingly different temporal regulation and tissue distribution. Using a nomenclature designed to stress important structural similarities of distinct gap junction gene products, the deduced polypeptides have been designated the Xenopus alpha 1 and alpha 2 gap junction proteins. The alpha 2 gap junction mRNA is a maternal transcript that disappears by the late gastrula stage. It is not detected in any organ of the adult except the ovary, and resides primarily, if not exclusively, in the oocytes and early embryos. The alpha 1 gap junction mRNA appears during organogenesis, and is detected in RNA from a wide variety of organs. It is also found in full-grown oocytes, but is rapidly degraded upon oocyte maturation, both in vivo and in vitro. The alpha 1 and alpha 2 mRNAs encode proteins with different degrees of amino acid sequence similarity to the predominant gap junction subunit of the mammalian heart (connexin 43). Together with our earlier report of a mid-embryonic (beta 1) gap junction mRNA, the results suggest that intercellular communication during oocyte growth and postfertilization development is a complex phenomenon involving the coordinated regulation of several genes. PMID:2155241

  16. 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...

  17. 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.

  18. 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

  19. Molecular basis of gap junctional communication in the CNS of the leech Hirudo medicinalis.

    PubMed

    Dykes, Iain M; Freeman, Fiona M; Bacon, Jonathan P; Davies, Jane A

    2004-01-28

    Gap junctions are intercellular channels that allow the passage of ions and small molecules between cells. In the nervous system, gap junctions mediate electrical coupling between neurons. Despite sharing a common topology and similar physiology, two unrelated gap junction protein families exist in the animal kingdom. Vertebrate gap junctions are formed by members of the connexin family, whereas invertebrate gap junctions are composed of innexin proteins. Here we report the cloning of two innexins from the leech Hirudo medicinalis. These innexins show a differential expression in the leech CNS: Hm-inx1 is expressed by every neuron in the CNS but not in glia, whereas Hm-inx2 is expressed in glia but not neurons. Heterologous expression in the paired Xenopus oocyte system demonstrated that both innexins are able to form functional homotypic gap junctions. Hm-inx1 forms channels that are not strongly gated. In contrast, Hm-inx2 forms channels that are highly voltage-dependent; these channels demonstrate properties resembling those of a double rectifier. In addition, Hm-inx1 and Hm-inx2 are able to cooperate to form heterotypic gap junctions in Xenopus oocytes. The behavior of these channels is primarily that predicted from the properties of the constituent hemichannels but also demonstrates evidence of an interaction between the two. This work represents the first demonstration of a functional gap junction protein from a Lophotrochozoan animal and supports the hypothesis that connexin-based communication is restricted to the deuterostome clade.

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

    PubMed Central

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

    2016-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.19510.001 PMID:27767956

  1. The role of gap junctions in inflammatory and neoplastic disorders (Review)

    PubMed Central

    Wong, Pui; Laxton, Victoria; Srivastava, Saurabh; Chan, Yin Wah Fiona; Tse, Gary

    2017-01-01

    Gap junctions are intercellular channels made of connexin proteins, mediating both electrical and biochemical signals between cells. The ability of gap junction proteins to regulate immune responses, cell proliferation, migration, apoptosis and carcinogenesis makes them attractive therapeutic targets for treating inflammatory and neoplastic disorders in different organ systems. Alterations in gap junction profile and expression levels are observed in hyperproliferative skin disorders, lymphatic vessel diseases, inflammatory lung diseases, liver injury and neoplastic disorders. It is now recognized that the therapeutic effects mediated by traditional pharmacological agents are dependent upon gap junction communication and may even act by influencing gap junction expression or function. Novel strategies for modulating the function or expression of connexins, such as the use of synthetic mimetic peptides and siRNA technology are considered. PMID:28098880

  2. 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.

  3. Novel model for the mechanisms of glutamate-dependent excitotoxicity: Role of neuronal gap junctions

    PubMed Central

    Belousov, Andrei B.

    2012-01-01

    In the mammalian central nervous system (CNS), coupling of neurons by gap junctions (electrical synapses) increases during early postnatal development, then decreases, but increases in the mature CNS following neuronal injury, such as ischemia, traumatic brain injury and epilepsy. Glutamate-dependent neuronal death also occurs in the CNS during development and neuronal injury, i.e., at the time when neuronal gap junction coupling is increased. Here, we review our recent studies on regulation of neuronal gap junction coupling by glutamate during development and injury and on the role of gap junctions in neuronal cell death. A novel model of the mechanisms of glutamate-dependent neuronal death is discussed, which includes neuronal gap junction coupling as a critical part of these mechanisms. PMID:22771704

  4. The role of gap junctions in inflammatory and neoplastic disorders (Review).

    PubMed

    Wong, Pui; Laxton, Victoria; Srivastava, Saurabh; Chan, Yin Wah Fiona; Tse, Gary

    2017-03-01

    Gap junctions are intercellular channels made of connexin proteins, mediating both electrical and biochemical signals between cells. The ability of gap junction proteins to regulate immune responses, cell proliferation, migration, apoptosis and carcinogenesis makes them attractive therapeutic targets for treating inflammatory and neoplastic disorders in different organ systems. Alterations in gap junction profile and expression levels are observed in hyperproliferative skin disorders, lymphatic vessel diseases, inflammatory lung diseases, liver injury and neoplastic disorders. It is now recognized that the therapeutic effects mediated by traditional pharmacological agents are dependent upon gap junction communication and may even act by influencing gap junction expression or function. Novel strategies for modulating the function or expression of connexins, such as the use of synthetic mimetic peptides and siRNA technology are considered.

  5. Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

    PubMed

    Gabashvili, A N; Baklaushev, V P; Grinenko, N F; Levinskii, A B; Mel'nikov, P A; Cherepanov, S A; Chekhonin, V P

    2015-05-01

    The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.

  6. Emergent Central Pattern Generator Behavior in Gap-Junction-Coupled Hodgkin-Huxley Style Neuron Model

    PubMed Central

    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 (IAHP) 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. PMID:23365558

  7. 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.

  8. Transfer of IP3 through gap junctions is critical, but not sufficient, for the spread of apoptosis

    PubMed Central

    Decrock, E; Krysko, D V; Vinken, M; Kaczmarek, A; Crispino, G; Bol, M; Wang, N; De Bock, M; De Vuyst, E; Naus, C C; Rogiers, V; Vandenabeele, P; Erneux, C; Mammano, F; Bultynck, G; Leybaert, L

    2012-01-01

    Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger. PMID:22117194

  9. 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

  10. Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in neuroectoderm. A study in vitro

    PubMed Central

    1988-01-01

    We studied the development of NCAM and gap junctional communication, and their mutual relationship in chick neuroectoderm in vitro. Expression of NCAM, as detected by monoclonal and polyclonal antibodies, and development of junctional communication, as detected by extensive cell-to-cell transfer of 400-500-D fluorescent tracers, occurred in cultures from stage-2 embryos onward. Both expressions presumably required primary induction. The differentiating cells formed discrete fields of expression on the second to third day in culture, with the NCAM fields coinciding with the junctional communication fields delineated by the tracers. Other neural differentiations developed in the following order: tetanus toxin receptors, neurofilament protein, and neurite outgrowth. Chronic treatment with antibody Fab fragments against NCAM interfered with the development of communication, suggesting that NCAM-mediated adhesion promotes formation of cell-to-cell channels. Temperature-sensitive mutant Rous sarcoma virus blocked (reversibly) communication and the subsequent development of neurofilament protein and neurites, but expression of NCAM continued. PMID:2834404

  11. 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.

  12. Peripheral inflammation augments gap junction-mediated coupling among satellite glial cells in mouse sympathetic ganglia.

    PubMed

    Hanani, Menachem; Caspi, Anna; Belzer, Vitali

    2010-02-01

    Intercellular coupling by gap junctions is one of the main features of glial cells, but very little is known about this aspect of satellite glial cells (SGCs) in sympathetic ganglia. We used the dye coupling method to address this question in both a prevertebral ganglion (superior mesenteric) and a paravertebral ganglion (superior cervical) of mice. We found that in control ganglia, the incidence of dye coupling among SGCs that form the envelope around a given neuron was 10-20%, and coupling between SGCs around different envelopes was rare (1.5-3%). The dye injections also provided novel information on the structure of SGCs. Following peripheral inflammation, both types of coupling were increased, but most striking was the augmentation of coupling between SGCs forming envelopes around different neurons, which rose by 8-14.6-fold. This effect appeared to be non-systemic, and was blocked by the gap junction blocker carbenoxolone. These changes in SGCs may affect signal transmission and processing in sympathetic ganglia.

  13. [Introduction to the structure and functions of junction communications or gap junctions].

    PubMed

    Rousset, B

    1996-01-01

    Cell-to-cell communication through gap junctions (GJ) represents a direct route of exchange of informations between neighboring cells within tissues and organs. GJ are formed from the assembly of a large number of channels that differ from the other known channels because they connect the cytoplasm of adjacent cells. The GJ channel is built from two parts: the connexons. A connexon inserted into the plasma membrane of a cell interacts with another connexon belonging to an adjacent cell. Connexons are composed of proteins with four transmembrane domains that are named connexins (Cx). Six Cx form a connexon. Cx belong to a protein family with 13 known members at present. Each Cx is defined by its molecular mass in kDa (ex: Cx32, Cx43...). A given cell type expresses one or several Cx. The cell to cell transfer of molecules through GJ channels exhibit a size selectivity; only molecules with a molecular mass lower than 1000 Da such as ions and second messengers freely pass through GJ. Depending on the Cx they are made of, GJ seem to differ somewhat in their permeability properties. Cell-to-cell communication via GJ is a regulated process. GJ channels can be either open or closed. GJ mediated cell-to-cell communication or junctional coupling can be detected and quantified by visualization of the cell to cell transfer of a fluorescent probe (such as Lucifer Yellow...) previously introduced in a single cell by microinjection. The presence of GJ channels can also be identified by recording the passage of an electric current between contiguous cells. GJ are involved in numerous fundamental biological processes from the embryonic development to the homeostasis in adult tissues and organs. GJ coordinate cell activities and sometimes synchronize cell behaviour. This is the case for the propagation of the excitation wave in the cardiac muscle and smooth muscle. GJ mediate metabolic cooperation between cells; they represent a way of supply of nutrients for tissues that are

  14. Local dynamics of gap-junction-coupled interneuron networks

    NASA Astrophysics Data System (ADS)

    Lau, Troy; Gage, Gregory J.; Berke, Joshua D.; Zochowski, Michal

    2010-03-01

    Interneurons coupled by both electrical gap-junctions (GJs) and chemical GABAergic synapses are major components of forebrain networks. However, their contributions to the generation of specific activity patterns, and their overall contributions to network function, remain poorly understood. Here we demonstrate, using computational methods, that the topological properties of interneuron networks can elicit a wide range of activity dynamics, and either prevent or permit local pattern formation. We systematically varied the topology of GJ and inhibitory chemical synapses within simulated networks, by changing connection types from local to random, and changing the total number of connections. As previously observed we found that randomly coupled GJs lead to globally synchronous activity. In contrast, we found that local GJ connectivity may govern the formation of highly spatially heterogeneous activity states. These states are inherently temporally unstable when the input is uniformly random, but can rapidly stabilize when the network detects correlations or asymmetries in the inputs. We show a correspondence between this feature of network activity and experimental observations of transient stabilization of striatal fast-spiking interneurons (FSIs), in electrophysiological recordings from rats performing a simple decision-making task. We suggest that local GJ coupling enables an active search-and-select function of striatal FSIs, which contributes to the overall role of cortical-basal ganglia circuits in decision-making.

  15. 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.

  16. 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

  17. Nonspecific effects of the gap junction blocker mefloquine on fast hippocampal network oscillations in the adult rat in vitro.

    PubMed

    Behrens, C J; Ul Haq, R; Liotta, A; Anderson, M L; Heinemann, U

    2011-09-29

    It has been suggested that gap junctions are involved in the synchronization during high frequency oscillations as observed during sharp wave-ripple complexes (SPW-Rs) and during recurrent epileptiform discharges (REDs). Ripple oscillations during SPW-Rs, possibly involved in memory replay and memory consolidation, reach frequencies of up to 200 Hz while ripple oscillations during REDs display frequencies up to 500 Hz. These fast oscillations may be synchronized by intercellular interactions through gap junctions. In area CA3, connexin 36 (Cx36) proteins are present and potentially sensitive to mefloquine. Here, we used hippocampal slices of adult rats to investigate the effects of mefloquine, which blocks Cx36, Cx43 and Cx50 gap junctions on both SPW-Rs and REDs. SPW-Rs were induced by high frequency stimulation in the CA3 region while REDs were recorded in the presence of the GABA(A) receptor blocker bicuculline (5 μM). Both, SPW-Rs and REDs were blocked by the gap junction blocker carbenoxolone. Mefloquine (50 μM), which did not affect stimulus-induced responses in area CA3, neither changed SPW-Rs nor superimposed ripple oscillations. During REDs, 25 and 50 μM mefloquine exerted only minor effects on the expression of REDs but significantly reduced the amplitude of superimposed ripples by ∼17 and ∼54%, respectively. Intracellular recordings of CA3 pyramidal cells revealed that mefloquine did not change their resting membrane potential and input resistance but significantly increased the afterhyperpolarization following evoked action potentials (APs) resulting in reduced probability of AP firing during depolarizing current injection. Similarly, mefloquine caused a reduction in AP generation during REDs. Together, our data suggest that mefloquine depressed RED-related ripple oscillations by reducing high frequency discharges and not necessarily by blocking electrical coupling.

  18. 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.

  19. 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

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

    PubMed

    Rash, J E; Kamasawa, N; Vanderpool, K G; Yasumura, T; O'Brien, J; Nannapaneni, S; Pereda, A E; Nagy, J I

    2015-01-29

    Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at "large myelinated club ending (LMCE)" synapses on Mauthner cells (M-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 connexin35 (Cx35) restricted to axon terminal hemiplaques and connexin34.7 (Cx34.7) restricted to apposing M-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 connexin36 (Cx36) on both sides, would require some other mechanism, such as differential phosphorylation of connexins on

  1. 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

  2. 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

  3. Identifying connexin expression and determining gap junction intercellular communication in rainbow trout cells.

    PubMed

    Hooper, Joshua; Poynter, Sarah J; DeWitte-Orr, Stephanie J

    2017-05-01

    Gap junctions are groups of membrane-bound channels that allow the passage of small molecules and ions between cells, permitting cell-cell communication. Because of their importance in cell homeostasis, gap junction presence and function were characterized in three commonly studied rainbow trout cell lines, namely RTgill-W1, RTgutGC, and RTG-2. Firstly, gap junction presence was determined by screening for gap junction protein alpha 7 and alpha 1 (GJA7 and GJA1) presence at the transcript level and GJA7 at the protein level. GJA7 was successfully identified at both the transcript and protein levels, and GJA1 was detected at the transcript level in all three cell lines. This is the first report of a GJA7 full-length transcript sequence in rainbow trout cells. Gap junction function, as determined by gap junction intercellular communication (GJIC), was examined using Lucifer yellow dye migration with the scrape and load technique; visualized by fluorescence microscopy. Phorbol 12-myristate 13-acetate (PMA), a gap junction inhibitor, was used to confirm the presence of functional gap junctions. Effects of serum deprivation on GJIC were also monitored; 24-h serum deprivation resulted in greater dye migration compared with 30-min serum deprivation. Both RTG-2 and RTgill-W1 showed significant dye migration that was inhibited by PMA while RTgutGC did not. Human foreskin fibroblast (HFF-1) cells were used as a positive control for gap junction presence and function. Taken together, our study shows that rainbow trout cells express connexin transcripts and proteins, and RTG-2 and, to a lesser extent, RTgill-W1 cells are able to perform GJIC.

  4. Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

    PubMed

    Zhang, Fang Fang; Morioka, Norimitsu; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-09-04

    Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. 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

  6. Conduction abnormalities and ventricular arrhythmogenesis: The roles of sodium channels and gap junctions

    PubMed Central

    Tse, Gary; Yeo, Jie Ming

    2015-01-01

    Ventricular arrhythmias arise from disruptions in the normal orderly sequence of electrical activation and recovery of the heart. They can be categorized into disorders affecting predominantly cellular depolarization or repolarization, or those involving action potential (AP) conduction. This article briefly discusses the factors causing conduction abnormalities in the form of unidirectional conduction block and reduced conduction velocity (CV). It then examines the roles that sodium channels and gap junctions play in AP conduction. Finally, it synthesizes experimental results to illustrate molecular mechanisms of how abnormalities in these proteins contribute to such conduction abnormalities and hence ventricular arrhythmogenesis, in acquired pathologies such as acute ischaemia and heart failure, as well as inherited arrhythmic syndromes. PMID:26839915

  7. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25150689

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

    PubMed

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

    2014-03-15

    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.

  10. 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

  11. Apparent AV junctional escape in Wenckebach AV block: markedly slow conduction through the slow AV pathway.

    PubMed

    Kinoshita, Shinji; Katoh, Takakazu; Hagisawa, Kohsuke; Fukushima, Tsutomu; Ikawa, Shinji

    2009-02-01

    We report here two cases of Wenckebach atrioventricular (AV) block in which apparent AV junctional escape was observed, but most likely resulted from markedly slow conduction through the slow pathway of dual AV junctional pathways. In these cases, it seems that a blocked P-wave was followed by an AV junctional escape beat. However, a blocked P-wave occasionally failed to be followed by an escape beat, and the RR interval containing the blocked P-wave was markedly longer than the above escape interval. In one case, apparent AV junctional escape beats with aberrant ventricular conduction were found, and QRS complexes of the same configuration were also found without the preceding ventricular pause. This strengthens the possibility that apparent AV junctional escape occurred because of markedly slow conduction through the slow AV pathway.

  12. CONNEXIN 43 AND BONE: NOT JUST A GAP JUNCTION PROTEIN

    PubMed Central

    Plotkin, Lilian I.

    2012-01-01

    Connexins are essential for the communication of cells among themselves and with their environment. Connexin hexamers assemble at the plasma membrane to form hemichannels that allow the exchange of cellular contents with the extracellular milieu. In addition, hemichannels expressed in neighboring cells align to form gap junction channels that mediate the exchange of contents among cells. Connexin 43 (Cx43) is the most abundant connexin expressed in bone cells and its deletion in all tissues leads to osteoblast dysfunction, as evidenced by reduced expression of osteoblast markers and delayed ossification. Moreover, Cx43 is essential for the survival of osteocytes; and mice lacking Cx43 in these cells exhibit increased prevalence of osteocyte apoptosis and empty lacunae in cortical bone. Work of several groups for the past few years has unveiled the role of Cx43 on the response of bone cells to a variety of stimuli. Thus, the preservation of the viability of osteoblasts and osteocytes by the anti-osteoporotic drugs bisphosphonates depends on Cx43 expression in vitro and in vivo. This survival effect does not require cell-to-cell communication and is mediated by unopposed hemichannels. Cx43 hemichannels are also required for the release of prostaglandins and ATP by osteocytes induced by mechanical stimulation in vitro. More recent evidence showed that the cAMP-mediated survival effect of parathyroid hormone (PTH) also requires Cx43 expression. Moreover, the hormone does not increase bone mineral content in mice haploinsufficient for Cx43 or lacking Cx43 in osteoblastic cells. Since inhibition of osteoblast apoptosis contributes, at least in part, to bone anabolism by PTH, the lack of response to the hormone might be due to the requirement of Cx43 for the effect of PTH on osteoblast survival. In summary, mounting evidence indicate that Cx43 is a key component of the intracellular machinery responsible for the transduction of signals in the skeleton in response to

  13. Nested transcripts of gap junction gene have distinct expression patterns.

    PubMed

    Zhang, Z; Curtin, K D; Sun, Y A; Wyman, R J

    1999-09-05

    The shaking B locus (shakB, or Passover) codes for structural molecules of gap junctions in Drosophila. This report describes the complex set of transcripts from the shakB locus. A nested set of five transcripts is described. The transcripts share 3' exons, but each has its own 5' exon. The transcripts are arrayed as a series in the genomic DNA stretching over 60 kb. The 5' end of each successive transcript lies further proximal on the chromosome. Each new transcript shares all the 3' exons with the one preceding it, but adds one or two more 5' exons. The different transcripts are expressed in a wide variety of locations in the nervous system and in non-neural tissues. Some tissues express more than one transcript, and the expression pattern of each is developmentally regulated. Within the adult central nervous system (CNS), these transcripts have an expression pattern that is restricted to the giant fiber system (GFS). The GFS is a small set of neurons which mediates the visually induced escape jump. shakB is required for function of the GFS electrical synapses. The transcript previously defined as active in the giant fiber is not, in fact, expressed in that cell. Instead, we find that another transcript, shakB(N3), and perhaps shakB(N4) as well, is expressed in the GFS; this transcript is not expressed elsewhere in the adult CNS. Two other transcripts, shakB(N1) and shakB(N2), are expressed in the optic lamina but not elsewhere in the CNS. This expression pattern explains the neurophysiological and behavioral defects in escape exhibited in mutants of shakB.

  14. 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.

  15. 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

  16. 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.

  17. 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

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

    PubMed

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

    2017-03-20

    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.

  19. 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,...

  20. 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,...

  1. Passover eliminates gap junctional communication between neurons of the giant fiber system in Drosophila. off.

    PubMed

    Sun, Y A; Wyman, R J

    1996-07-01

    The Passover-related gene family plays significant roles in cellular connectivity. Mutations in three family members from Drosophila and from Caenorhabditis elegans alter a few specific electrical synapses. The passage of cobalt between Drosophila neurons was used to assay the presence of gap junctional connections. The giant fiber in the wild type has specific gap junctional connections in the brain and in the thorax. In flies mutant for Passover, cobalt cannot pass into or out of the giant fiber in either the anterograde or the retrograde directions. A large number of other gap junctional connections remain unaffected. This demonstrates that the Passover gene is necessary for gap-junctional communication between the neurons of the Drosophila giant fiber system.

  2. 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

  3. Gap Junctions and Epileptic Seizures – Two Sides of the Same Coin?

    PubMed Central

    Volman, Vladislav; Perc, Matjaž; Bazhenov, Maxim

    2011-01-01

    Electrical synapses (gap junctions) play a pivotal role in the synchronization of neuronal ensembles which also makes them likely agonists of pathological brain activity. Although large body of experimental data and theoretical considerations indicate that coupling neurons by electrical synapses promotes synchronous activity (and thus is potentially epileptogenic), some recent evidence questions the hypothesis of gap junctions being among purely epileptogenic factors. In particular, an expression of inter-neuronal gap junctions is often found to be higher after the experimentally induced seizures than before. Here we used a computational modeling approach to address the role of neuronal gap junctions in shaping the stability of a network to perturbations that are often associated with the onset of epileptic seizures. We show that under some circumstances, the addition of gap junctions can increase the dynamical stability of a network and thus suppress the collective electrical activity associated with seizures. This implies that the experimentally observed post-seizure additions of gap junctions could serve to prevent further escalations, suggesting furthermore that they are a consequence of an adaptive response of the neuronal network to the pathological activity. However, if the seizures are strong and persistent, our model predicts the existence of a critical tipping point after which additional gap junctions no longer suppress but strongly facilitate the escalation of epileptic seizures. Our results thus reveal a complex role of electrical coupling in relation to epileptiform events. Which dynamic scenario (seizure suppression or seizure escalation) is ultimately adopted by the network depends critically on the strength and duration of seizures, in turn emphasizing the importance of temporal and causal aspects when linking gap junctions with epilepsy. PMID:21655239

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Inhibition of connexin43 gap junction channels by the endocrine disruptor ioxynil

    SciTech Connect

    Leithe, Edward; Kjenseth, Ane; Bruun, Jarle; Sirnes, Solveig; Rivedal, Edgar

    2010-08-15

    Gap junctions are intercellular plasma membrane domains containing channels that mediate transport of ions, metabolites and small signaling molecules between adjacent cells. Gap junctions play important roles in a variety of cellular processes, including regulation of cell growth and differentiation, maintenance of tissue homeostasis and embryogenesis. The constituents of gap junction channels are a family of trans-membrane proteins called connexins, of which the best-studied is connexin43. Connexin43 functions as a tumor suppressor protein in various tissue types and is frequently dysregulated in human cancers. The pesticide ioxynil has previously been shown to act as an endocrine disrupting chemical and has multiple effects on the thyroid axis. Furthermore, both ioxynil and its derivative ioxynil octanoate have been reported to induce tumors in animal bioassays. However, the molecular mechanisms underlying the possible tumorigenic effects of these compounds are unknown. In the present study we show that ioxynil and ioxynil octanoate are strong inhibitors of connexin43 gap junction channels. Both compounds induced rapid loss of connexin43 gap junctions at the plasma membrane and increased connexin43 degradation. Ioxynil octanoate, but not ioxynil, was found to be a strong activator of ERK1/2. The compounds also had different effects on the phosphorylation status of connexin43. Taken together, the data show that ioxynil and ioxynil octanoate are potent inhibitors of intercellular communication via gap junctions.

  9. 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

  10. Gap Junction Enhancer Potentiates Cytotoxicity of Cisplatin in Breast Cancer Cells.

    PubMed

    Ding, Ying; Nguyen, Thu Annelise

    2012-11-01

    Cisplatin is one of the most widely used anti-cancer drugs due to its ability to damage DNA and induce apoptosis. However, increasing reports of side effects and drug resistance indicate the limitation of cisplatin in cancer therapeutics. Recent studies showed that inhibition of gap junctions diminishes the cytotoxic effect and contributes to drug resistance. Therefore, identification of molecules that counteract gap junctional inhibition without decreasing the anti-cancer effect of cisplatin could be used in combinational treatment, potentiating cisplatin efficacy and preventing resistance. This study investigates the effects of combinational treatment of cisplatin and PQ1, a gap junction enhancer, in T47D breast cancer cells. Our results showed that combinational treatment of PQ1 and cisplatin increased gap junctional intercellular communication (GJIC) as well as expressions of connexins (Cx26, Cx32 and Cx43), and subsequently decreased cell viability. Ki67, a proliferation marker, was decreased by 75% with combinational treatment. Expressions of pro-apoptotic factors (cleaved caspase-3/-8/-9 and bax) were increased by the combinational treatment with PQ1 and cisplatin; whereas, the pro-survival factor, bcl-2, was decreased by the combinational treatment. Our study demonstrates for the first time that the combinational treatment with gap junction enhancers can counteract cisplatin induced inhibition of gap junctional intercellular communication and reduction of connexin expression, thereby increasing the efficacy of cisplatin in cancer cells.

  11. Cross regulation of intercellular gap junction communication and paracrine signaling pathways during organogenesis in Drosophila.

    PubMed

    Lechner, Hildegard; Josten, Frank; Fuss, Bernhard; Bauer, Reinhard; Hoch, Michael

    2007-10-01

    The spatial and temporal coordination of patterning and morphogenesis is often achieved by paracrine morphogen signals or by the direct coupling of cells via gap junctions. How paracrine signals and gap junction communication cooperate to control the coordinated behavior of cells and tissues is mostly unknown. We found that hedgehog signaling is required for the expression of wingless and of Delta/Notch target genes in a single row of boundary cells in the foregut-associated proventriculus organ of the Drosophila embryo. These cells coordinate the movement and folding of proventricular cells to generate a multilayered organ. hedgehog and wingless regulate gap junction communication by transcriptionally activating the innexin2 gene, which encodes a member of the innexin family of gap junction proteins. In innexin2 mutants, gap junction-mediated cell-to-cell communication is strongly reduced and the proventricular cell layers fail to fold and invaginate, similarly as in hedgehog or wingless mutants. We further found that innexin2 is required in a feedback loop for the transcriptional activation of the hedgehog and wingless morphogens and of Delta in the proventriculus primordium. We propose that the transcriptional cross regulation of paracrine and gap junction-mediated signaling is essential for organogenesis in Drosophila.

  12. Gap Junctions, Dendrites and Resonances: A Recipe for Tuning Network Dynamics

    PubMed Central

    2013-01-01

    Gap junctions, also referred to as electrical synapses, are expressed along the entire central nervous system and are important in mediating various brain rhythms in both normal and pathological states. These connections can form between the dendritic trees of individual cells. Many dendrites express membrane channels that confer on them a form of sub-threshold resonant dynamics. To obtain insight into the modulatory role of gap junctions in tuning networks of resonant dendritic trees, we generalise the “sum-over-trips” formalism for calculating the response function of a single branching dendrite to a gap junctionally coupled network. Each cell in the network is modelled by a soma connected to an arbitrary structure of dendrites with resonant membrane. The network is treated as a single extended tree structure with dendro-dendritic gap junction coupling. We present the generalised “sum-over-trips” rules for constructing the network response function in terms of a set of coefficients defined at special branching, somatic and gap-junctional nodes. Applying this framework to a two-cell network, we construct compact closed form solutions for the network response function in the Laplace (frequency) domain and study how a preferred frequency in each soma depends on the location and strength of the gap junction. PMID:23945377

  13. Gap junction networks in mushroom bodies participate in visual learning and memory in Drosophila

    PubMed Central

    Liu, Qingqing; Yang, Xing; Tian, Jingsong; Gao, Zhongbao; Wang, Meng; Li, Yan; Guo, Aike

    2016-01-01

    Gap junctions are widely distributed in the brains across species and play essential roles in neural information processing. However, the role of gap junctions in insect cognition remains poorly understood. Using a flight simulator paradigm and genetic tools, we found that gap junctions are present in Drosophila Kenyon cells (KCs), the major neurons of the mushroom bodies (MBs), and showed that they play an important role in visual learning and memory. Using a dye coupling approach, we determined the distribution of gap junctions in KCs. Furthermore, we identified a single pair of MB output neurons (MBONs) that possess a gap junction connection to KCs, and provide strong evidence that this connection is also required for visual learning and memory. Together, our results reveal gap junction networks in KCs and the KC-MBON circuit, and bring new insight into the synaptic network underlying fly’s visual learning and memory. DOI: http://dx.doi.org/10.7554/eLife.13238.001 PMID:27218450

  14. Reversible blockade of gap junctional communication by 2,3-butanedione monoxime in rat cardiac myocytes.

    PubMed

    Verrecchia, F; Hervé, J C

    1997-03-01

    2,3-Butanedione monoxime (BDM), a nucleophilic agent endowed with a "phosphatase-like" activity, is often used as a tool for investigating the effects of changes in phosphorylation level of protein constituents on membrane channel function. BDM produced a rapid, dosc-dependent, and reversible abolition of the cytosolic continuity existing between cells via gap junctional channels. The persistence of this effect when a nonhydrolyzable analogue of ATP [adenosine 5'-O-(3-thiotriphosphate) (ATP(gamma)S)] was introduced in the cytosol suggests that the acute suppressant effect of BDM was not due to dephosphorylation. However, the higher reversibility after BDM withdrawal in presence of ATP(gamma)S could signify that a protein-dephosphorylating activity gradually occurred during the oxime treatment. Junctional uncoupling took place even when the moderate increase in cytosolic Ca2+ concentration induced by BDM was prevented by ryanodine. These results are consistent with the model of dual mechanism of BDM action proposed for some other membrane channels, consisting of a quick channel block and a parallel slow inhibition, plausibly through dephosphorylation.

  15. A histone octamer blocks branch migration of a Holliday junction.

    PubMed Central

    Grigoriev, M; Hsieh, P

    1997-01-01

    The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome. PMID:9372946

  16. Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination.

    PubMed

    Personius, Kirkwood E; Chang, Qiang; Mentis, George Z; O'Donovan, Michael J; Balice-Gordon, Rita J

    2007-07-10

    During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40-/- mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40-/- mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.

  17. Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination

    PubMed Central

    Personius, Kirkwood E.; Chang, Qiang; Mentis, George Z.; O'Donovan, Michael J.; Balice-Gordon, Rita J.

    2007-01-01

    During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40−/− mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40−/− mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain. PMID:17609378

  18. The modulation of gap-junctional intercellular communication by lipid rafts.

    PubMed

    Defamie, Norah; Mesnil, Marc

    2012-08-01

    Lipid rafts are specific microdomains of plasma membrane which are enriched in cholesterol and sphingolipids. These domains seem to favour the interactions of particular proteins and the regulation of signalling pathways in the cells. Recent data have shown that among the proteins, which are preferentially localized in lipid rafts, are connexins that are the structural proteins of gap junctions. Since gap junctional intercellular communication is involved in various cellular processes and pathologies such as cancer, we were interested to review the various observations concerning this specific localization of connexins in lipid rafts and its consequences on gap junctional intercellular communication capacity. In particular, we will focus our discussion on the role of the lipid raft-connexin connection in cancer progression. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

  19. 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

  20. Trafficking of gap junction channels at a vertebrate electrical synapse in vivo.

    PubMed

    Flores, Carmen E; Nannapaneni, Srikant; Davidson, Kimberly G V; Yasumura, Thomas; Bennett, Michael V L; Rash, John E; Pereda, Alberto E

    2012-02-28

    Trafficking and turnover of transmitter receptors required to maintain and modify the strength of chemical synapses have been characterized extensively. In contrast, little is known regarding trafficking of gap junction components at electrical synapses. By combining ultrastructural and in vivo physiological analysis at identified mixed (electrical and chemical) synapses on the goldfish Mauthner cell, we show here that gap junction hemichannels are added at the edges of GJ plaques where they dock with hemichannels in the apposed membrane to form cell-cell channels and, simultaneously, that intact junctional regions are removed from centers of these plaques into either presynaptic axon or postsynaptic dendrite. Moreover, electrical coupling is readily modified by intradendritic application of peptides that interfere with endocytosis or exocytosis, suggesting that the strength of electrical synapses at these terminals is sustained, at least in part, by fast (in minutes) turnover of gap junction channels. A peptide corresponding to a region of the carboxy terminus that is conserved in Cx36 and its two teleost homologs appears to interfere with formation of new gap junction channels, presumably by reducing insertion of hemichannels on the dendritic side. Thus, our data indicate that electrical synapses are dynamic structures and that their channels are turned over actively, suggesting that regulated trafficking of connexons may contribute to the modification of gap junctional conductance.

  1. 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

  2. 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.

  3. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas.

    PubMed

    Wu, Jie; Gao, Ming; Rice, Stephen G; Tsang, Candy; Beggs, John; Turner, Dharshaun; Li, Guohui; Yang, Bo; Xia, Kunkun; Gao, Fenfei; Qiu, Shenfeng; Liu, Qiang; Kerrigan, John F

    2016-06-01

    Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients. Copyright © 2016 The Authors. Published by

  4. Gap junctions in the ventral hippocampal-medial prefrontal pathway are involved in anxiety regulation.

    PubMed

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

    2014-11-19

    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. Copyright © 2014 the authors 0270-6474/14/3415679-10$15.00/0.

  5. 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.

  6. 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

  7. Computational model of erratic arrhythmias in a cardiac cell network: the role of gap junctions.

    PubMed

    Casaleggio, Aldo; Hines, Michael L; Migliore, Michele

    2014-01-01

    Cardiac morbidity and mortality increases with the population age. To investigate the underlying pathological mechanisms, and suggest new ways to reduce clinical risks, computational approaches complementing experimental and clinical investigations are becoming more and more important. Here we explore the possible processes leading to the occasional onset and termination of the (usually) non-fatal arrhythmias widely observed in the heart. Using a computational model of a two-dimensional network of cardiac cells, we tested the hypothesis that an ischemia alters the properties of the gap junctions inside the ischemic area. In particular, in agreement with experimental findings, we assumed that an ischemic episode can alter the gap junctions of the affected cells by reducing their average conductance. We extended these changes to include random fluctuations with time, and modifications in the gap junction rectifying conductive properties of cells along the edges of the ischemic area. The results demonstrate how these alterations can qualitatively give an account of all the main types of non-fatal arrhythmia observed experimentally, and suggest how premature beats can be eliminated in three different ways: a) with a relatively small surgical procedure, b) with a pharmacological reduction of the rectifying conductive properties of the gap-junctions, and c) by pharmacologically decreasing the gap junction conductance. In conclusion, our model strongly supports the hypothesis that non-fatal arrhythmias can develop from post-ischemic alteration of the electrical connectivity in a relatively small area of the cardiac cell network, and suggests experimentally testable predictions on their possible treatments.

  8. Netrin and Frazzled regulate presynaptic gap junctions at a Drosophila giant synapse.

    PubMed

    Orr, Brian O; Borgen, Melissa A; Caruccio, Phyllis M; Murphey, Rodney K

    2014-04-16

    Netrin and its receptor, Frazzled, dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the presynaptic terminal. In Netrin mutant animals, the synaptic coupling between a giant interneuron and the "jump" motor neuron was weakened and dye coupling between these two neurons was severely compromised or absent. In cases in which Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses and dye coupling between the giant fiber (GF) and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap junctions with antibodies to Shaking-B (ShakB) Innexin, they were significantly decreased or absent in the presynaptic terminal of the mutant GF. Frazzled loss of function mutants exhibited similar defects in synaptic transmission, dye coupling, and gap junction localization. These data are the first to show that Netrin and Frazzled regulate the placement of gap junctions presynaptically at a synapse.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Gap Junction Contributions to the Goldfish Electroretinogram at the Photopic Illumination Level

    PubMed Central

    Kim, Doh-Yeon

    2012-01-01

    Understanding how the b-wave of the electroretinogram (ERG) is generated by full-field light stimulation is still a challenge in visual neuroscience. To understand more about the origin of the b-wave, we studied the contributions of gap junctions to the ERG b-wave. Many types of retinal neurons are connected to similar and different neighboring neurons through gap junctions. The photopic (cone-dominated) ERG, stimulated by a small light beam, was recorded from goldfish (Carassius auratus) using a corneal electrode. Data were obtained before and after intravitreal injection of agents into the eye under a photopic illumination level. Several agents were used to affect gap junctions, such as dopamine D1 and D2 receptor agonists and antagonists, a nitric oxide (NO) donor, a nitric oxide synthase (NOS) inhibitor, the gap junction blocker meclofenamic acid (MFA), and mixtures of these agents. The ERG b-waves, which were enhanced by MFA, sodium nitroprusside (SNP), SKF 38393, and sulpiride, remained following application of a further injection of a mixture with MFA. The ERG b-waves decreased following NG-nitro-L-arginine methyl ester (L-NAME), SCH 23390, and quinpirole administration but were enhanced by further injection of a mixture with MFA. These results indicate that gap junction activity influences b-waves of the ERG related to NO and dopamine actions. PMID:22802705

  14. Gap junctional communication during human trophoblast differentiation: influence of human chorionic gonadotropin.

    PubMed

    Cronier, L; Bastide, B; Hervé, J C; Délèze, J; Malassiné, A

    1994-07-01

    During pregnancy, the trophoblast develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. As the exchange of molecules through gap junctions is considered to play a role in the control of cell and tissue differentiation, the cell to cell diffusion of a fluorescent dye was investigated in human trophoblastic cells differentiating in culture. The fluorescence recovery after photobleaching technique was used to estimate the transfer of 6-carboxyfluorescein from contiguous cellular elements into photobleached cells. Fluorescence recovery follows a slow exponential time course when the cell to cell exchange process is rate limited by the presence of gap junctional channels between contiguous cells, contrasting with a much faster step-like course in the case of fusion of the plasma membranes. In the presence of 10% fetal calf serum, Percoll-purified cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 24-48 h after plating. During this in vitro differentiation, fluorescence recoveries after photobleaching with a time course typical for gap junctions were observed between aggregated cytotrophoblastic cells, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous syncytiotrophoblasts. The maximum percentage of gap junctional coupling occurs on the fourth day. This fluorescence recovery is attributed to the diffusion of dye through gap junctions, because it can be interrupted by exposure to a known junctional uncoupler (3 mM heptanol). The effects of hCG on this gap junctional communication during trophoblast differentiation were investigated. In the presence of 500 mIU/ml hCG in the culture medium, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days of culture vs. 4 days in control medium. Moreover, the diffusion rate constant k (the inverse value of the time constant measured on recovery curves) was

  15. Effects of chronic gap junction conduction-enhancing antiarrhythmic peptide GAP-134 administration on experimental atrial fibrillation in dogs.

    PubMed

    Laurent, Gabriel; Leong-Poi, Howard; Mangat, Iqwal; Moe, Gordon W; Hu, Xudong; So, Petsy Pui-Sze; Tarulli, Emidio; Ramadeen, Andrew; Rossman, Eric I; Hennan, James K; Dorian, Paul

    2009-04-01

    Abnormal intercellular communication caused by connexin dysfunction may contribute to atrial fibrillation (AF). The present study assessed the effect of the gap junction conduction-enhancing antiarrhythmic peptide GAP-134 on AF inducibility and maintenance in a dog model of atrial cardiomyopathy. Twenty-four dogs subject to simultaneous atrioventricular pacing (220 bpm for 14 days) were randomly assigned to placebo treatment (PACED-CTRL; 12 dogs) or oral GAP-134 (2.9 mg/kg BID; PACED-GAP-134; 12 dogs) starting on day 0. UNPACED-CTRL (4 dogs) and UNPACED-GAP-134 (4 dogs) served as additional control groups. Change in left atrial (LA) systolic area from baseline to 14 days was calculated using transoesophageal echocardiography. At 14 days, animals underwent an open-chest electrophysiological study. PACED-CTRL dogs (versus UNPACED-CTRL) had a shorter estimated LA wavelength (8.0+/-1.4 versus 24.4+/-2.5 cm, P<0.05) and a greater AF vulnerability (mean AF duration, 1588+/-329 versus 25+/-34 seconds, P<0.05). Oral GAP-134 had no effect on AF vulnerability in UNPACED dogs. Compared with PACED-CTRL dogs, PACED-GAP-134 dogs had a longer estimated LA wavelength (10.2+/-2.8 versus 8.0+/-1.4 cm, respectively, P<0.05). Oral GAP-134 did not significantly reduce AF inducibility or maintenance in the entire group of 24 PACED dogs; in a subgroup of dogs (n=11) with less than 100% increase in LA systolic area, oral GAP-134 reduced AF induction from 100% to 40% and mean AF duration from 1737+/-120 to 615+/-280 seconds (P<0.05). Oral GAP-134 reduces pacing-induced decrease in LA wavelength and appears to attenuate AF vulnerability in dogs with less atrial mechanical remodeling. Gap junction modulation may affect AF in some circumstances.

  16. 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.

  17. 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.

  18. Protein kinase C-dependent regulation of connexin43 gap junctions and hemichannels.

    PubMed

    Alstrom, Jette Skov; Stroemlund, Line Waring; Nielsen, Morten Schak; MacAulay, Nanna

    2015-06-01

    Connexin43 (Cx43) generates intercellular gap junction channels involved in, among others, cardiac and brain function. Gap junctions are formed by the docking of two hemichannels from neighbouring cells. Undocked Cx43 hemichannels can upon different stimuli open towards the extracellular matrix and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current knowledge of protein kinase C (PKC)-dependent regulation of Cx43 and discuss the divergent results.

  19. Gap junctions enhancer combined with Vaughan Williams class III antiarrhythmic drugs, a promising antiarrhythmic method?

    PubMed

    Li, Lian-dong; Zhang, Cun-tai; Ruan, Lei; Ni, Ming-ke; Quan, Xiao-qing

    2011-01-01

    Arrhythmias is one of the leading causes of death in the world. Current antiarrhythmic drugs are limited by unsatisfactory efficacy and adverse effects such as proarrhythmias. Reentry mechanism plays an important role in persistence of arrhythmias. Reentry can only continue when reentry path-length is longer than cardiac wavelength which is equal to the product of conduction velocity (CV) and effective refractory period (ERP). Gap junctions uncoupling is associated with proarrhythmic CV slowing and transmural dispersion of repolarization (TDR) increasing in many cardiac diseases. Vaughan Williams class III antiarrhythmic drugs prolong ERP with an augmented TDR which is the main mechanism of the proarrhythmic effects. Gap junctions enhancer can augment CV and diminish TDR. As a result, gap junctions enhancer combined with class III drugs may be a promising antiarrhythmic method.

  20. Closing the gap in the Andreev spectrum in a three-terminal superconducting junction

    NASA Astrophysics Data System (ADS)

    Padurariu, Ciprian; Melin, Régis; Jonckheere, Thibaut; Rech, Jérôme; Martin, Thierry; Feinberg, Denis; Douçot, Benoît; Nazarov, Yuli

    2015-03-01

    Quasiclassical circuit theory is used to investigate transport in a mesoscopic junction with three superconducting terminals. Our study reveals the closing of the gap in the Andreev spectrum for a wide range of phase-biases and transparencies, in agreement with previous work. In this regime a superconducting current flows in the junction, while the proximity mini-gap is closed. The corresponding parameter region is studied systematically, both analytically in the low transparency limit and numerically. We provide a microscopic explanation for the closing of the gap in terms of multiple pair processes that correlate the superconducting currents flowing between different pairs of terminals. We show that multi-terminal superconducting junctions provide unique opportunities for applications in quantum devices based on Josephson and/or Majorana physics.

  1. Size and selectivity of gap junction channels formed from different connexins.

    PubMed

    Veenstra, R D

    1996-08-01

    Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of approximately 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.

  2. Spatially resolved gap closing in single Josephson junctions constructed on Bi2Te3 surface

    NASA Astrophysics Data System (ADS)

    Pang, Yuan; Wang, Junhua; Lyu, Zhaozheng; Yang, Guang; Fan, Jie; Liu, Guangtong; Ji, Zhongqing; Jing, Xiunian; Yang, Changli; Lu, Li

    2016-11-01

    Full gap closing is a prerequisite for hosting Majorana zero modes in Josephson junctions on the surface of topological insulators. Previously, we have observed direct experimental evidence of gap closing in Josephson junctions constructed on Bi2Te3 surface. In this paper we report further investigations on the position dependence of gap closing as a function of magnetic flux in single Josephson junctions constructed on Bi2Te3 surface. Project supported by the National Basic Research Program of China (Grant Nos. 2009CB929101 and 2011CB921702), the National Natural Science Foundation of China (Grant Nos. 91221203, 11174340, 11174357, 91421303, and 11527806), and the Strategic Priority Research Program B of the Chinese Academy of Sciences (Grant No. XDB07010100).

  3. Gap junctions in C. elegans: Their roles in behavior and development.

    PubMed

    Hall, David H

    2016-06-13

    The nematode Caenorhabditis elegans utilizes gap junctions in different fashions in virtually all of its cells. This model animal has a surprisingly large number of innexin genes within its genome, and many nematode cell types can express multiple innexins at once, leading to the formation of diverse junction types and enough redundancy to limit the effect of single gene knockdowns on animal development or behavioral phenotypes. Here we review the general properties of these junctions, their expression patterns, and their known roles in tissue development and in the animal's connectome. This article is protected by copyright. All rights reserved.

  4. Gap junctions in C. elegans: Their roles in behavior and development

    PubMed Central

    2016-01-01

    ABSTRACT The nematode Caenorhabditis elegans utilizes gap junctions in different fashions in virtually all of its cells. This model animal has a surprisingly large number of innexin genes within its genome, and many nematode cell types can express multiple innexins at once, leading to the formation of diverse junction types and enough redundancy to limit the effect of single gene knockdowns on animal development or behavioral phenotypes. Here, we review the general properties of these junctions, their expression patterns, and their known roles in tissue development and in the animal's connectome. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 587–596, 2017 PMID:27294317

  5. Targeted deletion of Aqp4 promotes the formation of astrocytic gap junctions.

    PubMed

    Katoozi, Shirin; Skauli, Nadia; Rahmani, Soulmaz; Camassa, Laura M A; Boldt, Henning B; Ottersen, Ole P; Amiry-Moghaddam, Mahmood

    2017-05-27

    Aquaporin-4 (AQP4) is the predominant water channel in the brain and is expressed in high density in astrocytes. By fluxing water along osmotic gradients, AQP4 contributes to brain volume and ion homeostasis. Here we ask whether deletion of Aqp4 leads to upregulation of the gap junctional proteins connexin-43 (Cx43) and connexin-30 (Cx30). These molecules couple adjacent astrocytes to each other and allow water and ions to redistribute within the astrocyte syncytium. Immunogold analysis of parietal cortex and hippocampus showed that the number of gap junctions per capillary profile is increased in AQP4 knockout (AQP4 KO) mice. The most pronounced changes were observed for Cx43 in hippocampus where the number of connexin labeled gap junctions increased by 100% following AQP4 KO. Western blot analysis of whole tissue homogenates showed no change in the amount of Cx43 or Cx30 protein after AQP4 KO. However, AQP4 KO led to a significant increase in the amount of Cx43 in a Triton X-100 insoluble fraction. This fraction is associated with connexin assembly into gap junctional plaques in the plasma membrane. In line with our immunoblot data, RT-qPCR showed no significant increase in Cx43 and Cx30 mRNA levels after AQP4 KO. Our findings suggest that AQP4 KO leads to increased aggregation of Cx43 into gap junctions and provide a putative mechanistic basis for the enhanced tracer coupling in hippocampi of AQP4 KO mice. The increased number of gap junctions in AQP4 deficient mice may explain why Aqp4 deletion has rather modest effects on brain volume and K(+) homeostasis.

  6. Gap junctional communication between the satellite cells of rat dorsal root ganglia.

    PubMed

    Sakuma, E; Wang, H J; Asai, Y; Tamaki, D; Amano, K; Mabuchi, Y; Herbert, D C; Soji, T

    2001-06-01

    Many studies have described the ultrastructure of the dorsal root ganglia in various embryonic and adult animals, but in spite of the efforts of many investigators the functional role of the satellite cells in this tissue is not clearly understood. In this study, we discuss the function of this cell type based on the concept of cell-to-cell interaction through gap junctions. Five male 60 day-old Wistar strain rats were used. All animals were anesthetized with pentobarbital and perfused with glutaraldehyde fixative, then the dorsal root ganglia in levels L4, L5 and L6 were taken from each rat. After postosmication, the specimens were prepared for observation by transmission electron microscopy. All nerve cells were completely surrounded by satellite cell cytoplasmic expansions. The boundaries between adjacent nerve cells and satellite cells were complicated due to the presence of perikaryal projections of nerve cells. Gap junctions which showed the typical trilamellar structure of plasma membranes were found mainly between satellite cell processes belonging to the same nerve cell. On the other hand, some gap junctions were found between the satellite cell projections belonging to different nerve cells. The size of the gap junctions ranged from 300 to 400 nm. No gap junctions were associated with the plasma membrane of any nerve cell. In conclusion, only satellite cells can share free transcellular exchange of cytoplasmic molecules such as ions, amino acids, sugars and several second messengers including cAMP and inositol 1,4,5-triphosphate by way of gap junctions in dorsal root ganglia.

  7. Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

    SciTech Connect

    Shinotsuka, Takanori; Yasui, Masato; Nuriya, Mutsuo

    2014-02-07

    Highlights: • Astrocytes exhibit characteristic changes in [Ca{sup 2+}]{sub i} under OGD. • Astrocytic [Ca{sup 2+}]{sub i} increase is synchronized with a neuronal anoxic depolarization. • Gap junctional couplings protect neurons as well as astrocytes during OGD. - Abstract: Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca{sup 2+}]{sub i}) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca{sup 2+}]{sub i} oscillations followed by larger and sustained [Ca{sup 2+}]{sub i} increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca{sup 2+}]{sub i} increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca{sup 2+}]{sub i} increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca{sup 2+}]{sub i} increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage

  8. 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

  9. Gap junction intercellular communication: a review of a potential platform to modulate craniofacial tissue engineering.

    PubMed

    Rossello, Ricardo A; Kohn, David H

    2009-02-01

    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. Copyright 2008 Wiley Periodicals, Inc.

  10. Theory of heterotic superconductor-insulator-superconductor Josephson junctions between single- and multiple-gap superconductors.

    PubMed

    Ota, Yukihiro; Machida, Masahiko; Koyama, Tomio; Matsumoto, Hideki

    2009-06-12

    Using the functional integral method, we construct a theory of heterotic superconductor-insulator-superconductor Josephson junctions between one- and two-gap superconductors. The theory predicts the presence of in-phase and out-of-phase collective oscillation modes of superconducting phases. The former corresponds to the Josephson plasma mode whose frequency is drastically reduced for +/- s-wave symmetry, and the latter is a counterpart of Leggett's mode in Josephson junctions. We also reveal that the critical current and the Fraunhofer pattern strongly depend on the symmetry type of the two-gap superconductor.

  11. Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

    PubMed Central

    1993-01-01

    The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states. PMID:8301264

  12. Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.

    PubMed

    Carystinos, G D; Katabi, M M; Laird, D W; Galipeau, J; Chan, H; Alaoui-Jamali, M A; Batist, G

    1999-01-01

    The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

  13. Role of hippocampal CA1 area gap junction channels on morphine state-dependent learning.

    PubMed

    Beheshti, Siamak; Hosseini, Seyyed Akbar Mir Seyyed; Noorbakhshnia, Maryam; Eivani, Mehdi

    2014-12-15

    Morphine produces a state dependent learning. The hippocampus is involved in this kind of learning. Gap junctions (GJs) are involved in some of the effects of morphine and exist in different areas of the hippocampus. We investigated the effects of blocking GJ channels of the hippocampal CA1 area, by means of pre-test bilateral injection of carbenoxolone (CBX), on morphine state dependent learning, using a passive avoidance task. Post-training subcutaneous administrations of morphine (0.5, 2.5, 5 and 7.5 mg/kg) dose-dependently impaired memory retrieval. Pre-test administration of morphine (0.5, 2.5, 5 and 7.5 mg/kg) induced a state-dependent retrieval of the memory acquired under post-training morphine influence. Pre-test injections of CBX (25, 75 and 150 nM) dose dependently prevented memory retrieval by post-training (7.5 mg/kg) and pre-test (0.5, 2.5, 5, 7.5 mg/kg) injections of morphine. The results suggest that intercellular coupling via GJ channels of the hippocampal CA1 area modulates morphine state dependent learning.

  14. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

    PubMed

    Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

    2009-08-14

    Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

  15. Modulation of Asymmetric Flux in Heterotypic Gap Junctions by Pore Shape, Particle Size and Charge.

    PubMed

    Mondal, Abhijit; Sachse, Frank B; Moreno, Alonso P

    2017-01-01

    Gap junction channels play a vital role in intercellular communication by connecting cytoplasm of adjoined cells through arrays of channel-pores formed at the common membrane junction. Their structure and properties vary depending on the connexin isoform(s) involved in forming the full gap junction channel. Lack of information on the molecular structure of gap junction channels has limited the development of computational tools for single channel studies. Currently, we rely on cumbersome experimental techniques that have limited capabilities. We have earlier reported a simplified Brownian dynamics gap junction pore model and demonstrated that variations in pore shape at the single channel level can explain some of the differences in permeability of heterotypic channels observed in in vitro experiments. Based on this computational model, we designed simulations to study the influence of pore shape, particle size and charge in homotypic and heterotypic pores. We simulated dye diffusion under whole cell voltage clamping. Our simulation studies with pore shape variations revealed a pore shape with maximal flux asymmetry in a heterotypic pore. We identified pore shape profiles that match the in silico flux asymmetry results to the in vitro results of homotypic and heterotypic gap junction formed out of Cx43 and Cx45. Our simulation results indicate that the channel's pore-shape established flux asymmetry and that flux asymmetry is primarily regulated by the sizes of the conical and/or cylindrical mouths at each end of the pore. Within the set range of particle size and charge, flux asymmetry was found to be independent of particle size and directly proportional to charge magnitude. While particle charge was vital to creating flux asymmetry, charge magnitude only scaled the observed flux asymmetry. Our studies identified the key factors that help predict asymmetry. Finally, we suggest the role of such flux asymmetry in creating concentration imbalances of messenger

  16. Connexin26 regulates assembly and maintenance of cochlear gap junction macromolecular complex for normal hearing

    NASA Astrophysics Data System (ADS)

    Kamiya, Kazusaku; Fukunaga, Ichiro; Hatakeyama, Kaori; Ikeda, Katsuhisa

    2015-12-01

    Hereditary deafness affects about 1 in 2000 children and GJB2 gene mutation is most frequent cause for this disease in the world. GJB2 encodes connexin26 (Cx26), a component in cochlear gap junction. Recently, we found macromolecular change of gap junction plaques with two different types of Cx26 mutation as major classification of clinical case, one is a model of dominant negative type, Cx26R75W+ and the other is conditional gene deficient mouse, Cx26f/fP0Cre as a model for insufficiency of gap junction protein [6]. Gap junction composed mainly of Cx26 and Cx30 in wild type mice formed large planar gap junction plaques (GJP). In contrast, Cx26R75W+ and Cx26f/fP0Cre showed fragmented small round GJPs around the cell border. In Cx26f/fP0Cre, some of the cells with Cx26 expression due to their cellular mosaicism showed normal large GJP with Cx26 and Cx30 only at the cell junction site between two Cx26 positive cells. These indicate that bilateral Cx26 expressions from both adjacent cells are essential for the formation of the cochlear linear GJP, and it is not compensated by other cochlear Connexins such as Connexin30. In the present study, we demonstrated a new molecular pathology in most common hereditary deafness with different types of Connexin26 mutations, and this machinery can be a new target for drag design of hereditary deafness.

  17. Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear, stacked linear, curvilinear, oval, and annular junctions.

    PubMed

    Shapiro, F

    1997-10-01

    There is a marked variability in the conformation of bone cell gap junctions in newborn murine cortical bone as defined by transmission electron microscopy (TEM). Studies were done in newborn BALB/c mouse and Sprague-Dawley rat femurs and tibias. Femoral and tibial cortices were dissected into 1 mm3 fragments and prepared in standardized fashion using modified Karnovsky fixation, 7.5% EDTA decalcification, 1% osmium tetroxide-sym collidine buffer with 1% lanthanum nitrate postfixation, Epon resin, 60 nm sections, lead citrate/uranyl acetate staining, and examination at 60 kV. Previous TEM descriptions of bone junctions have, with rare exceptions, noted only isolated linear or mildly curvilinear structures. In this study we noted gap junctional shapes on thin-section TEM preparations of osteoblasts and osteocytes to be extremely variable and complex encompassing linear, curvilinear, stacked linear, oval, and annular conformations. Multiple observations revealed linear gap junctions linking surface osteoblast cell bodies; linear, curvilinear, stacked linear, and oval junctions linking osteoblast processes in osteoid; linear and curvilinear junctions where cell processes joined with osteocyte cell bodies and each of the five conformations linking osteocyte processes within canaliculi. The annular junctions were found within osteoblast and osteocyte cytoplasm and in osteocyte cell processes within canaliculi. The annular junctions are intracellular, degenerating structures which appear as ultrastructural markers of gap junction involution. The more complex shapes reported here must be considered in (1) interpreting quantitative studies using freeze-fracture replicas, thin sections, and confocal microscopy immunolabeled junction connexin-43 components and (2) assessing gap junction biogenesis and turnover. 3-D reconstruction of bone junctions will enhance our understanding of these complex conformations.

  18. Voltage-controlled switching and thermal effects in VO{sub 2} nano-gap junctions

    SciTech Connect

    Joushaghani, Arash; Jeong, Junho; Stewart Aitchison, J.; Poon, Joyce K. S.; Paradis, Suzanne; Alain, David

    2014-06-02

    Voltage-controlled switching in lateral VO{sub 2} nano-gap junctions with different gap lengths and thermal properties was investigated. The effect of Joule heating on the phase transition was found to be strongly influenced by the device geometry, the contact material, and the current. Our results indicate that the VO{sub 2} phase transition was likely initiated electronically, which was sometimes followed by a secondary thermally induced transition.

  19. Gap Junctions Link Regular-Spiking and Fast-Spiking Interneurons in Layer 5 Somatosensory Cortex.

    PubMed

    Hatch, Robert J; Mendis, G Dulini C; Kaila, Kai; Reid, Christopher A; Petrou, Steven

    2017-01-01

    Gap junctions form electrical synapses that modulate neuronal activity by synchronizing action potential (AP) firing of cortical interneurons (INs). Gap junctions are thought to form predominantly within cortical INs of the same functional class and are therefore considered to act within discrete neuronal populations. Here, we challenge that view and show that the probability of electrical coupling is the same within and between regular-spiking (RS) and fast-spiking (FS) cortical INs in 16-21 days old mice. Firing properties of these two populations were distinct from other INs types including neurogliaform and low-threshold spiking (LTS) cells. We also demonstrate that pre-junctional APs can depolarize post-junctional neurons and increase the probability of firing. Our findings of frequent gap junction coupling between functionally distinct IN subtypes suggest that cortical IN networks are much more extensive and heterogeneous than previously thought. This may have implications on mechanisms ranging from cognitive functions to modulation of pathological states in epilepsy and other neurological disorders.

  20. The action of v-src on gap junctional permeability is modulated by pH

    PubMed Central

    1990-01-01

    The product of the viral src gene (v-src) is the protein tyrosine kinase pp60v-src. Among the known consequences of pp60v-src activity is the reduction in permeability of gap junctions, an effect that is counteracted by the calcium antagonist TMB-8 (8-N,N-[diethylamino]octyl- 3,4,5-trimethoxybenzoate). We show here that a decrease in intracellular pH (pHi) also counteracts the v-src effect: junctional permeability of cells containing active v-src kinase rose with decreasing pHi in the range 7.15 to 6.75, whereas junctional permeability of cells containing inactive v-src kinase or no v-src at all was insensitive to pH in that range. Low pH also counteracted the known action of diacylglycerol on junction, but only when pp60v-src kinase was inactive. Immunoblots of whole-cell lysates using an antibody against phosphotyrosine show that phosphorylation on tyrosine of at least one cellular protein, specific for pp60v-src kinase activity, was reduced by low pH but not by TMB-8. These results suggest that TMB-8 does not inhibit v-src action on junctional permeability by interfering with tyrosine phosphorylation of a protein crucial for closure of gap junction channels, but that the inhibition by low pH may be via this mechanism. PMID:2157717

  1. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity.

    PubMed

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-12

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

  2. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity

    PubMed Central

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-01

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we utilized a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on GFP-positive astrocytes in acute brain slices from GFAP-GFP mice at 3 or 7 days following S. aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced EtBr uptake that could be blocked by several pharmacological inhibitors including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide 10Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 post-infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (GLT and GLAST) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component. PMID:21228152

  3. 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.

  4. A functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide.

    PubMed

    Geletu, Mulu; Guy, Stephanie; Firth, Kevin; Raptis, Leda

    2014-10-18

    In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner. Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

  5. 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

  6. 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
    ...

  7. Electrical signal transmission in a bone cell network: the influence of a discrete gap junction

    NASA Technical Reports Server (NTRS)

    Zhang, D.; Weinbaum, S.; Cowin, S. C.

    1998-01-01

    A refined electrical cable model is formulated to investigate the role of a discrete gap junction in the intracellular transmission of electrical signals in an electrically coupled system of osteocytes and osteoblasts in an osteon. The model also examines the influence of the ratio q between the membrane's electrical time constant and the characteristic time of pore fluid pressure, the circular, cylindrical geometry of the osteon, and key simplifying assumptions in our earlier continuous cable model (see Zhang, D., S. C. Cowin, and S. Weinbaum. Electrical signal transmission and gap junction regulation in a bone cell network: A cable model for an osteon. Ann. Biomed. Eng. 25:379-396, 1997). Using this refined model, it is shown that (1) the intracellular potential amplitude at the osteoblastic end of the osteonal cable retains the character of a combination of a low-pass and a high-pass filter as the corner frequency varies in the physiological range; (2) the presence of a discrete gap junction near a resting osteoblast can lead to significant modulation of the intracellular potential and current in the osteoblast for measured values of the gap junction coupling strength; and (3) the circular, cylindrical geometry of the osteon is well simulated by the beam analogy used in Zhang et al.

  8. Gap Junctions and Hemichannels in Signal Transmission, Function and Development of Bone

    PubMed Central

    Batra, Nidhi; Kar, Rekha; Jiang, Jean X.

    2012-01-01

    Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2 kDa including small ions, metabolites, ATP, prostaglandin and IP3. The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. PMID:21963408

  9. Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes.

    PubMed

    Suswillo, Rosemary F L; Javaheri, Behzad; Rawlinson, Simon C F; Dowthwaite, Gary P; Lanyon, Lance E; Pitsillides, Andrew A

    2017-01-01

    Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity. Copyright © 2017 The Authors Cell Biochemistry and Function published by John Wiley & Sons Ltd.

  10. 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...

  11. 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
    ...

  12. 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...

  13. 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.

  14. Intrinsic Islet Heterogeneity and Gap Junction Coupling Determine Spatiotemporal Ca2+ Wave Dynamics

    PubMed Central

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

    2014-01-01

    Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca2+]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 [Ca2+]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 [Ca2+]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 [Ca2+]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. PMID:25468351

  15. A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

    PubMed Central

    Geletu, Mulu; Guy, Stephanie; Firth, Kevin; Raptis, Leda

    2014-01-01

    In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition. PMID:25350637

  16. Electrical signal transmission in a bone cell network: the influence of a discrete gap junction

    NASA Technical Reports Server (NTRS)

    Zhang, D.; Weinbaum, S.; Cowin, S. C.

    1998-01-01

    A refined electrical cable model is formulated to investigate the role of a discrete gap junction in the intracellular transmission of electrical signals in an electrically coupled system of osteocytes and osteoblasts in an osteon. The model also examines the influence of the ratio q between the membrane's electrical time constant and the characteristic time of pore fluid pressure, the circular, cylindrical geometry of the osteon, and key simplifying assumptions in our earlier continuous cable model (see Zhang, D., S. C. Cowin, and S. Weinbaum. Electrical signal transmission and gap junction regulation in a bone cell network: A cable model for an osteon. Ann. Biomed. Eng. 25:379-396, 1997). Using this refined model, it is shown that (1) the intracellular potential amplitude at the osteoblastic end of the osteonal cable retains the character of a combination of a low-pass and a high-pass filter as the corner frequency varies in the physiological range; (2) the presence of a discrete gap junction near a resting osteoblast can lead to significant modulation of the intracellular potential and current in the osteoblast for measured values of the gap junction coupling strength; and (3) the circular, cylindrical geometry of the osteon is well simulated by the beam analogy used in Zhang et al.

  17. 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

  18. Reversible structure transition in gap junction under Ca++ control seen by high-resolution electron microscopy.

    PubMed

    Wrigley, N G; Brown, E; Chillingworth, R K

    1984-01-01

    Deoxycholate-extracted rat liver gap junction was studied by high-resolution low-dose electron microscopy. Communicating channels between two adjoining cells supposedly form along the common axis of two apposed hexameric trans-membrane protein assemblies. These double hexamers are often arranged in large plaques on an ordered hexagonal net (8-9 nm lattice constant) and seem able to undergo structural alteration as a possible permeability control mechanism. Calcium is widely reported to uncouple gap junction, and we observed this alteration on exposure to Ca++ down to 10(-4) M concentration. When EGTA was added at matching concentrations, the alteration was reversible several times over one hour, but with considerable variability. It was imaged in the absence of any negative stain to avoid ionic and other complications. The resulting lack of contrast plus low-dose "shot" noise required digital Fourier filtering and reconstruction, but no detail was recovered below 1.8 nm. In other experiments with negative stain at neutral pH, gap junction connexons were apparently locked in the "closed" configuration and no transition could be induced. However, recovery of repeating detail to nearly 1.0 nm was possible, reproducibly showing a fine connective matrix between connexons . Whether this was formed by unfolded portions of the 28,000-dalton gap junction protein is not known, but its existence could explain the observed lattice invariance during the connexon structural transition.

  19. 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...

  20. 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

  1. 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...

  2. Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes

    PubMed Central

    Suswillo, Rosemary F.L.; Rawlinson, Simon C.F.; Dowthwaite, Gary P.; Lanyon, Lance E.; Pitsillides, Andrew A.

    2017-01-01

    Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity. PMID:28083967

  3. 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.

  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. 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

  6. Atomic structure of the innexin-6 gap junction channel determined by cryo-EM

    PubMed Central

    Oshima, Atsunori; Tani, Kazutoshi; Fujiyoshi, Yoshinori

    2016-01-01

    Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families. PMID:27905396

  7. 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.

  8. Oxaliplatin enhances gap junction-mediated coupling in cell cultures of mouse trigeminal ganglia.

    PubMed

    Poulsen, Jeppe Nørgaard; Warwick, Rebekah; Duroux, Meg; Hanani, Menachem; Gazerani, Parisa

    2015-08-01

    Communications between satellite glial cells and neighboring neurons within sensory ganglia may contribute to neuropathic and inflammatory pain. To elucidate the role of satellite glial cells in chemotherapy-induced pain, we examined the effects of oxaliplatin on the gap junction-mediated coupling between these cells. We also examined whether the gap junction blocker, carbenoxolone, can reverse the coupling. Primary cultures of mice trigeminal ganglia, 24-48h after cell isolation, were used. Satellite glial cells were injected with Lucifer yellow in the presence or absence of oxaliplatin (60 μM). In addition, the effect of carbenoxolone (100 μM) on coupling, and the expression of connexin 43 proteins were evaluated. Dye coupling between adjacent satellite glial cells was significantly increased (2.3-fold, P<0.05) following a 2h incubation with oxaliplatin. Adding carbenoxolone to the oxaliplatin-treated cultures reversed oxaliplatin-evoked coupling to baseline (P<0.05). Immunostaining showed no difference between expression of connexin 43 in control and oxaliplatin-treated cultures. Our findings indicated that oxaliplatin-increased gap junction-mediated coupling between satellite glial cells in primary cultures of mouse trigeminal ganglia, and carbenoxolone reversed this effect. Hence, it is proposed that increased gap junction-mediated coupling was seen between satellite glial cells in TG. This observation together with our previous data obtained from a behavioral study suggests that this phenomenon might contribute to chemotherapy-induced nociception following oxaliplatin treatment.

  9. 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...

  10. Regulation of gap junctional intercellular communication by TCDD in HMEC and MCF-7 breast cancer cells

    SciTech Connect

    Gakhar, Gunjan Schrempp, Diane Nguyen, Thu Annelise

    2009-03-01

    Previous studies suggest that many neoplastic tissues exhibit a decrease in gap junctional intercellular communication (GJIC). Many hydrocarbons and organochlorine compounds are environmental pollutants known to be carcinogenic. The effect of an organochlorine compound, TCDD, on GJIC in human breast cell lines has not been established. In the present study, we showed that TCDD causes an inhibition in the gap junctional activity in MCF-7 (breast cancer cells). In MCF-7 cells, an increase in the phosphorylated form of gap junctional protein, connexin 43 (Cx43), and PKC {alpha} was seen in the presence of TCDD. Gap junctional plaque formation was significantly decreased in MCF-7 cells in the presence of TCDD. Immunoprecipitation studies of PKC {alpha} showed that TCDD caused a significant 40% increase in the phosphorylated Cx43 in MCF-7 cells. TCDD also modulated the translocation of PKC {alpha} from the cytosol to the membrane and caused a 2-fold increase in the PKC {alpha} activity at 50 nM TCDD in MCF-7 cells. Calphostin C, an inhibitor of PKC {alpha}, showed a significant inhibition of PKC {alpha} activity in the presence of TCDD. Furthermore, TCDD also caused a decrease in the gap junctional activity and Cx43 protein in human mammary epithelial cells (HMEC). However, we observed a shift in the Cx43 plaques towards the perinuclear membrane in the presence of TCDD by confocal microscopy and Western blot. Overall, these results conclude that TCDD decreases GJIC by phosphorylating Cx43 via PKC {alpha} signaling pathway in MCF-7 cells; however, TCDD decreases the GJIC by affecting the localization of Cx43 in HMEC. These new findings elucidate the differential mode of effect of TCDD in the downregulation of GJIC in HMEC and MCF-7 cells.

  11. Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

    PubMed

    Xia, Cong-Yuan; Chu, Shi-Feng; Zhang, Shuai; Gao, Yan; Ren, Qian; Lou, Yu-Xia; Luo, Piao; Tian, Man-Tong; Wang, Zhi-Qi; Du, Guo-Hua; Tomioka, Yoshihisa; Yamakuni, Tohru; Zhang, Yi; Wang, Zhen-Zhen; Chen, Nai-Hong

    2017-08-17

    Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50μM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10μM) or fluoxetine (1μM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1μM and 10μM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  12. Sustained inhibition of rat myometrial gap junctions and contractions by lindane

    PubMed Central

    Loch-Caruso, Rita K; Criswell, Kay A; Grindatti, Carmen M; Brant, Kelly A

    2003-01-01

    Background Gap junctions increase in size and abundance coincident with parturition, forming an intercellular communication network that permits the uterus to develop the forceful, coordinated contractions necessary for delivery of the fetus. Lindane, a pesticide used in the human and veterinary treatment of scabies and lice as well as in agricultural applications, inhibits uterine contractions in vitro, inhibits myometrial gap junctions, and has been associated with prolonged gestation length in rats. The aim of the present study was to investigate whether brief exposures to lindane would elicit sustained inhibition of rat uterine contractile activity and myometrial gap junction intercellular communication. Methods To examine effects on uterine contraction, longitudinal uterine strips isolated from late gestation (day 20) rats were exposed to lindane in muscle baths and monitored for changes in spontaneous phasic contractions during and after exposure to lindane. Lucifer yellow dye transfer between myometrial cells in culture was used to monitor gap junction intercellular communication. Results During a 1-h exposure, 10 micro M and 100 micro M lindane decreased peak force and frequency of uterine contraction but 1 micro M lindane did not. After removal of the exposure buffer, contraction force remained significantly depressed in uterine strips exposed to 100 micro M lindane, returning to less than 50% basal levels 5 h after cessation of lindane exposure. In cultured myometrial myocytes, significant sustained inhibition of Lucifer yellow dye transfer was observed 24 h after lindane exposures as brief as 10 min and as low as 0.1 micro M lindane. Conclusion Brief in vitro exposures to lindane have long-term effects on myometrial functions that are necessary for parturition, inhibiting spontaneous phasic contractions in late gestation rat uterus and gap junction intercellular communication in myometrial cell cultures. PMID:14567758

  13. 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.

  14. Adenosine and dopamine receptors co-regulate photoreceptor coupling via gap junction phosphorylation in mouse retina

    PubMed Central

    Li, Hongyan; Zhang, Zhijing; Blackburn, Michael R.; Wang, Steven W.; Ribelayga, Christophe P.; O’Brien, John

    2013-01-01

    Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibits adenylyl cyclase (AC) and reduces PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to co-regulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately down-regulated expression of D4R and AC type I mRNA. We conclude that adenosine and dopamine co-regulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function. PMID:23407968

  15. The gap junction cellular internet: connexin hemichannels enter the signalling limelight.

    PubMed

    Evans, W Howard; De Vuyst, Elke; Leybaert, Luc

    2006-07-01

    Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology.

  16. The gap junction cellular internet: connexin hemichannels enter the signalling limelight

    PubMed Central

    Evans, W. Howard; De Vuyst, Elke; Leybaert, Luc

    2006-01-01

    Cxs (connexins), the protein subunits forming gap junction intercellular communication channels, are transported to the plasma membrane after oligomerizing into hexameric assemblies called connexin hemichannels (CxHcs) or connexons, which dock head-to-head with partner hexameric channels positioned on neighbouring cells. The double membrane channel or gap junction generated directly couples the cytoplasms of interacting cells and underpins the integration and co-ordination of cellular metabolism, signalling and functions, such as secretion or contraction in cell assemblies. In contrast, CxHcs prior to forming gap junctions provide a pathway for the release from cells of ATP, glutamate, NAD+ and prostaglandin E2, which act as paracrine messengers. ATP activates purinergic receptors on neighbouring cells and forms the basis of intercellular Ca2+ signal propagation, complementing that occuring more directly via gap junctions. CxHcs open in response to various types of external changes, including mechanical, shear, ionic and ischaemic stress. In addition, CxHcs are influenced by intracellular signals, such as membrane potential, phosphorylation and redox status, which translate external stresses to CxHc responses. Also, recent studies demonstrate that cytoplasmic Ca2+ changes in the physiological range act to trigger CxHc opening, indicating their involvement under normal non-pathological conditions. CxHcs not only respond to cytoplasmic Ca2+, but also determine cytoplasmic Ca2+, as they are large conductance channels, suggesting a prominent role in cellular Ca2+ homoeostasis and signalling. The functions of gap-junction channels and CxHcs have been difficult to separate, but synthetic peptides that mimic short sequences in the Cx subunit are emerging as promising tools to determine the role of CxHcs in physiology and pathology. PMID:16761954

  17. Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation*

    PubMed Central

    Wang, Ke; Gu, Sumin; Yin, Xinye; Weintraub, Susan T.; Hua, Zichun; Jiang, Jean X.

    2012-01-01

    The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging. PMID:22418432

  18. 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.

  19. 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.

  20. C-reactive protein, sodium azide, and endothelial connexin43 gap junctions.

    PubMed

    Wang, Hsueh-Hsiao; Yeh, Hung-I; Wang, Chi-Young; Su, Cheng-Huang; Wu, Yih-Jer; Tseng, Yuen-Yi; Lin, Yi-Chun; Tsai, Cheng-Ho

    2010-04-01

    We investigated the effect of C-reactive protein (CRP) and sodium azide (NaN(3)) on endothelial Cx43 gap junctions. Human aortic endothelial cells (HAEC) were treated with (a) detoxified CRP, (b) detoxified dialyzed CRP, (c) detoxified dialyzed CRP plus NaN(3), (d) NaN(3), or (e) dialyzed NaN(3). The concentration of CRP in all preparations was fixed to 25 microg/ml and that of NaN(3) in the preparations of (c) to (e) was equivalent to that contained in the 25 microg/ml CRP purchased commercially. The results showed that both the expression of Cx43 protein and gap junctional communication function post-48-h incubation were reduced and inhibited by the detoxified CRP, NaN(3), or detoxified dialyzed CRP plus NaN(3), but not by the detoxified dialyzed CRP or dialyzed NaN(3). Reverse transcription-polymerase chain reaction analysis of cells treated for 72 h also showed a pattern of transcriptional regulation essentially the same as that for the proteins. We concluded that CRP does not have a significant effect on Cx43 gap junctions of HAEC, but NaN(3) inhibited the viability of cells and downregulate their junctions.

  1. Massive Dirac fermion transport in a gapped graphene-based magnetic tunnel junction

    NASA Astrophysics Data System (ADS)

    Soodchomshom, Bumned; Tang, I.-Ming; Hoonsawat, Rassmidara

    2009-08-01

    The spin transport in a graphene-based magnetic (NG/ferromagnetic barrier (FB)/NG) tunnel junction with the graphene sheet being grown on a SiC substrate is investigated. Zhou et al. [Nat. Mater. 6 (2007) 770] has shown that in these epitaxial grown graphene sheets, the electrons behave like massive relativistic particles with an energy gap of 2 Δ∼260 meV opening up in the energy spectrum of the massive relativistic electron. Basing on assumption that gap in graphene can occur under the influence of the magnetic field, we find that in the case of thick ferromagnetic graphene barriers, the electronic gap causes the barrier to behave as a strong insulator when the gate potential is in the range 400-130 meV< V G<400+130 meV (and the energy level Ef∼400 meV above Dirac point). For these values of V G, the spin polarization of the junction is P(%)∼100% except for V G= E f, where it is P(%)=0%. The current of the junction, for thick FB, can be rapidly switched from a 100% spin up current to a 100% spin down current by small variation of V G from V G< E f to V G> E f , the features of a perfect spin filtering electronic junction.

  2. Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.

    PubMed

    Quist, A P; Rhee, S K; Lin, H; Lal, R

    2000-03-06

    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 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 beta-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.

  3. Estradiol Reduces Connexin43 Gap Junctions in the Uterus during Adenomyosis in Cows.

    PubMed

    Korzekwa, A J; Łupicka, M; Socha, B M; Szczepańska, A A

    2016-09-01

    Adenomyosis is defined as the presence of glandular foci external to the endometrium of the uterus, either in the myometrium or/and perimetrium, depending on the progress of this dysfunction. To date, we showed that steroids secretion and prolactin expression and proliferative processes are disturbed during uterine adenomyosis in cows. During endometriosis in eutopic endometrium in women, gap junctions are down regulated. The transmembrane gap junction protein, connexin (Cx43) is necessary for endometrial morphological, biochemical and angiogenic functions. The aim of this study is recognition of adenomyosis etiology by determination of the role of Cx43 in this process. Immunolocalization and comparison of Cx43 mRNA and protein expression in healthy (N=9) and adenomyotic uterine tissue (N=9), and Cx43 mRNA expression (real time PCR) in uterine stromal - myometrium co-culture under 24-hour stimulation with 17-beta estradiol (10-7M) isolated from healthy (N=5) and adenomyotic (N=5) cows were determined. Cx43 was localized in healthy and adenomyotic uteri. mRNA and protein expression was down-regulated in uterine tissue in adenomyotic compared with healthy cows (p<0.05). Estradiol stimulated Cx43 mRNA expression in myometrial cell culture and co-culture of stromal and myometrial cells in adenomyotic compared with healthy cows (p<0.05). In summary, down-regulation of Cx43 expression in the junction zone might play an important role in pathogenesis of adenomyosis. Estradiol modulates gap junctions during adenomyosis.

  4. Connexin43 Mutation Causes Heterogeneous Gap Junction Loss and Sudden Infant Death

    PubMed Central

    Van Norstrand, David W.; Asimaki, Angeliki; Rubinos, Clio; Dolmatova, Elena; Srinivas, Miduturu; Tester, David J.; Saffitz, Jeffrey E.; Duffy, Heather S.; Ackerman, Michael J.

    2012-01-01

    Background An estimated 10-15% of sudden infant death syndrome (SIDS) may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1-encoded gap junction channel protein connexin43 (Cx43) is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death. Methods and Results GJA1 open reading frame mutational analysis was performed using PCR, DHPLC, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole cell patch-clamp studies were performed to determine functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white male and a 3-month-old white female, respectively. Analysis of the E42K victim’s parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in over 1000 ethnic-matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation and S272P demonstrated wildtype junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss-of-function not rescued by wildtype. Moreover, the E42K victim cardiac tissue demonstrated a mosaic immunostaining pattern for Cx43 protein. Conclusions This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-Cx43 demonstrated a trafficking-independent reduction in junctional coupling in vitro as well as demonstrating a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of Cx43-associated sudden death. PMID:22179534

  5. 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

  6. Two Drosophila Innexins Are Expressed in Overlapping Domains and Cooperate to Form Gap-Junction Channels

    PubMed Central

    Stebbings, Lucy A.; Todman, Martin G.; Phelan, Pauline; Bacon, Jonathan P.; Davies, Jane A.

    2000-01-01

    Members of the innexin protein family are structural components of invertebrate gap junctions and are analogous to vertebrate connexins. Here we investigate two Drosophila innexin genes, Dm-inx2 and Dm-inx3 and show that they are expressed in overlapping domains throughout embryogenesis, most notably in epidermal cells bordering each segment. We also explore the gap-junction–forming capabilities of the encoded proteins. In paired Xenopus oocytes, the injection of Dm-inx2 mRNA results in the formation of voltage-sensitive channels in only ∼ 40% of cell pairs. In contrast, Dm-Inx3 never forms channels. Crucially, when both mRNAs are coexpressed, functional channels are formed reliably, and the electrophysiological properties of these channels distinguish them from those formed by Dm-Inx2 alone. We relate these in vitro data to in vivo studies. Ectopic expression of Dm-inx2 in vivo has limited effects on the viability of Drosophila, and animals ectopically expressing Dm-inx3 are unaffected. However, ectopic expression of both transcripts together severely reduces viability, presumably because of the formation of inappropriate gap junctions. We conclude that Dm-Inx2 and Dm-Inx3, which are expressed in overlapping domains during embryogenesis, can form oligomeric gap-junction channels. PMID:10888681

  7. Protein kinase Cδ-mediated phosphorylation of Connexin43 gap junction channels causes movement within gap junctions followed by vesicle internalization and protein degradation.

    PubMed

    Cone, Angela C; Cavin, Gabriel; Ambrosi, Cinzia; Hakozaki, Hiroyuki; Wu-Zhang, Alyssa X; Kunkel, Maya T; Newton, Alexandra C; Sosinsky, Gina E

    2014-03-28

    Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.

  8. 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.

  9. Role of gap junction channel in the development of beat-to-beat action potential repolarization variability and arrhythmias.

    PubMed

    Magyar, Janos; Banyasz, Tamas; Szentandrassy, Norbert; Kistamas, Kornel; Nanasi, Peter P; Satin, Jonathan

    2015-01-01

    The short-term beat-to-beat variability of cardiac action potential duration (SBVR) occurs as a random alteration of the ventricular repolarization duration. SBVR has been suggested to be more predictive of the development of lethal arrhythmias than the action potential prolongation or QT prolongation of ECG alone. The mechanism underlying SBVR is not completely understood but it is known that SBVR depends on stochastic ion channel gating, intracellular calcium handling and intercellular coupling. Coupling of single cardiomyocytes significantly decreases the beat-to-beat changes in action potential duration (APD) due to the electrotonic current flow between neighboring cells. The magnitude of this electrotonic current depends on the intercellular gap junction resistance. Reduced gap junction resistance causes greater electrotonic current flow between cells, and reduces SBVR. Myocardial ischaemia (MI) is known to affect gap junction channel protein expression and function. MI increases gap junction resistance that leads to slow conduction, APD and refractory period dispersion, and an increase in SBVR. Ultimately, development of reentry arrhythmias and fibrillation are associated post-MI. Antiarrhythmic drugs have proarrhythmic side effects requiring alternative approaches. A novel idea is to target gap junction channels. Specifically, the use of gap junction channel enhancers and inhibitors may help to reveal the precise role of gap junctions in the development of arrhythmias. Since cell-to-cell coupling is represented in SBVR, this parameter can be used to monitor the degree of coupling of myocardium.

  10. Sustained rhythmic activity in gap-junctionally coupled networks of model neurons depends on the diameter of coupled dendrites

    PubMed Central

    Gansert, Juliane; Golowasch, Jorge; Nadim, Farzan

    2008-01-01

    Gap junctions are known to be important for many network functions such as synchronization of activity and the generation of waves and oscillations. Gap junctions have also been proposed to be essential for the generation of early embryonic activity. We have previously shown that the amplitude of electrical signals propagating across gap-junctionally coupled passive cables is maximized at a unique diameter. This suggests that threshold-dependent signals may propagate through gap junctions for a finite range of diameters around this optimal value. Here we examine the diameter dependence of action potential propagation across model networks of dendro-dendritically coupled neurons. The neurons in these models have passive soma and dendrites and an action potential generating axon. We show that propagation of action potentials across gap junctions occurs only over a finite range of dendritic diameters and that propagation delay depends on this diameter. Additionally, in networks of gap-junctionally coupled neurons, rhythmic activity can emerge when closed loops (re-entrant paths) occur but again only for a finite range of dendrite diameters. The frequency of such rhythmic activity depends on the length of the path and the dendrite diameter. For large networks of randomly coupled neurons, we find that the re-entrant paths that underlie rhythmic activity also depend on dendrite diameter. These results underline the potential importance of dendrite diameter as a determinant of network activity in gap-junctionally coupled networks, such as network rhythms that are observed during early nervous system development. PMID:17913989

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

    PubMed Central

    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. PMID:27023006

  12. Connexin Hemichannels and Gap Junction Channels Are Differentially Influenced by Lipopolysaccharide and Basic Fibroblast Growth Factor

    PubMed Central

    De Vuyst, Elke; Decrock, Elke; De Bock, Marijke; Yamasaki, Hiroshi; Naus, Christian C.; Evans, W. Howard

    2007-01-01

    Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell–cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies. PMID:17079735

  13. Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor.

    PubMed

    De Vuyst, Elke; Decrock, Elke; De Bock, Marijke; Yamasaki, Hiroshi; Naus, Christian C; Evans, W Howard; Leybaert, Luc

    2007-01-01

    Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.

  14. A versatile optical junction using photonic band-gap guidance and self collimation

    SciTech Connect

    Gupta, Man Mohan; Medhekar, Sarang

    2014-09-29

    We show that it is possible to design two photonic crystal (PC) structures such that an optical beam of desired wavelength gets guided within the line defect of the first structure (photonic band gap guidance) and the same beam gets guided in the second structure by self-collimation. Using two dimensional simulation of a design made of the combination of these two structures, we propose an optical junction that allows for crossing of two optical signals of same wavelength and same polarization with very low crosstalk. Moreover, the junction can be operated at number of frequencies in a wide range. Crossing of multiple beams with very low cross talk is also possible. The proposed junction should be important in future integrated photonic circuits.

  15. Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells.

    PubMed

    Talaverón, Rocío; Fernández, Paola; Escamilla, Rosalba; Pastor, Angel M; Matarredona, Esperanza R; Sáez, Juan C

    2015-01-01

    The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain.

  16. 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

  17. Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells

    PubMed Central

    Talaverón, Rocío; Fernández, Paola; Escamilla, Rosalba; Pastor, Angel M.; Matarredona, Esperanza R.; Sáez, Juan C.

    2015-01-01

    The postnatal subventricular zone (SVZ) lining the walls of the lateral ventricles contains neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. Communication via gap junctions between cells in the SVZ is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. SVZ NPCs can be expanded in vitro in the form of neurospheres that can be used for transplantation purposes after brain injury. We have previously reported that neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when they are implanted after mechanical injury. To analyze functionality of NPC-glial cell gap junctions we performed dye coupling experiments in co-cultures of SVZ NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, Cx45 and pannexin 1 (Panx1). Dye coupling experiments revealed that gap junctional communication occurred among neurosphere cells (incidence of coupling: 100%). Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap junction blocker. Altogether, these results suggest the existence of functional hemichannels and gap junction channels in postnatal SVZ neurospheres. In addition, they demonstrate that SVZ-derived NPCs can establish functional gap junctions with astrocytes or microglia. Therefore, cell-cell communication via gap junctions and hemichannels with host glial cells might subserve a role in the functional integration of NPCs after implantation in the damaged brain. PMID:26528139

  18. A rapid and sensitive assay of intercellular coupling by voltage imaging of gap junction networks

    PubMed Central

    2013-01-01

    Background A variety of mechanisms that govern connexin channel gating and permeability regulate coupling in gap junction networks. Mutations in connexin genes have been linked to several pathologies, including cardiovascular anomalies, peripheral neuropathy, skin disorders, cataracts and deafness. Gap junction coupling and its patho–physiological alterations are commonly assayed by microinjection experiments with fluorescent tracers, which typically require several minutes to allow dye transfer to a limited number of cells. Comparable or longer time intervals are required by fluorescence recovery after photobleaching experiments. Paired electrophysiological recordings have excellent time resolution but provide extremely limited spatial information regarding network connectivity. Results Here, we developed a rapid and sensitive method to assay gap junction communication using a combination of single cell electrophysiology, large–scale optical recordings and a digital phase–sensitive detector to extract signals with a known frequency from Vf2.1.Cl, a novel fluorescent sensor of plasma membrane potential. Tests performed in HeLa cell cultures confirmed that suitably encoded Vf2.1.Cl signals remained confined within the network of cells visibly interconnected by fluorescently tagged gap junction channels. We used this method to visualize instantly intercellular connectivity over the whole field of view (hundreds of cells) in cochlear organotypic cultures from postnatal mice. A simple resistive network model reproduced accurately the spatial dependence of the electrical signals throughout the cellular network. Our data suggest that each pair of cochlear non−sensory cells of the lesser epithelial ridge is coupled by ~1500 gap junction channels, on average. Junctional conductance was reduced by 14% in cochlear cultures harboring the T5M mutation of connexin30, which induces a moderate hearing loss in connexin30T5M/T5M knock–in mice, and by 91% in cultures from

  19. Modulation of Asymmetric Flux in Heterotypic Gap Junctions by Pore Shape, Particle Size and Charge

    PubMed Central

    Mondal, Abhijit; Sachse, Frank B.; Moreno, Alonso P.

    2017-01-01

    Gap junction channels play a vital role in intercellular communication by connecting cytoplasm of adjoined cells through arrays of channel-pores formed at the common membrane junction. Their structure and properties vary depending on the connexin isoform(s) involved in forming the full gap junction channel. Lack of information on the molecular structure of gap junction channels has limited the development of computational tools for single channel studies. Currently, we rely on cumbersome experimental techniques that have limited capabilities. We have earlier reported a simplified Brownian dynamics gap junction pore model and demonstrated that variations in pore shape at the single channel level can explain some of the differences in permeability of heterotypic channels observed in in vitro experiments. Based on this computational model, we designed simulations to study the influence of pore shape, particle size and charge in homotypic and heterotypic pores. We simulated dye diffusion under whole cell voltage clamping. Our simulation studies with pore shape variations revealed a pore shape with maximal flux asymmetry in a heterotypic pore. We identified pore shape profiles that match the in silico flux asymmetry results to the in vitro results of homotypic and heterotypic gap junction formed out of Cx43 and Cx45. Our simulation results indicate that the channel's pore-shape established flux asymmetry and that flux asymmetry is primarily regulated by the sizes of the conical and/or cylindrical mouths at each end of the pore. Within the set range of particle size and charge, flux asymmetry was found to be independent of particle size and directly proportional to charge magnitude. While particle charge was vital to creating flux asymmetry, charge magnitude only scaled the observed flux asymmetry. Our studies identified the key factors that help predict asymmetry. Finally, we suggest the role of such flux asymmetry in creating concentration imbalances of messenger

  20. A gap-junction-mediated signal, rather than an external paracrine factor, predominates during meiotic induction in isolated mouse oocytes.

    PubMed

    Downs, S M

    2001-02-01

    This study was carried out to compare the possible role of a secreted paracrine factor versus that of a gap-junction-transmitted signal in mediating meiotic induction in isolated mouse oocytes from PMSG-primed, immature mice. In the first set of experiments, oocyte-cumulus cell complexes (OCC) were pretreated for 3 h with 2 mM dbcAMP or FSH, washed, and the oocytes then cultured for 17-18 h in 40 microl drops containing either 300 microM dbcAMP or 4 mM hypoxanthine (HX). Each set of pretreated oocytes was cultured under three different conditions: (1) intact cumulus-cell-enclosed oocytes (CEO); (2) denuded oocytes (DO), cultured alone after removal of cumulus cells; and (3) co-cultured cumulus cells and oocytes (CC/DO), where the cumulus cells were removed in the same drop with a mouth-operated pipette and cultured alongside the oocytes. When pretreated with high dbcAMP or FSH, maturation was stimulated in CEO when cultured in either inhibitor (by 41.4-53.7%). Pretreatment failed to affect the maturation rate in DO. DO maturation was not altered appreciably by co-cultured cumulus cells when arrest was maintained with dbcAMP. However, an increase in maturation of 21-23% was observed in CC/DO in the HX-containing cultures that was not dependent on prior treatment with a meiosis-inducing stimulus. When DO were co-cultured with intact, FSH-treated OCC, there was no evidence of a positive factor secreted by the stimulated complexes, despite the fact that oocytes within the OCC were induced to resume maturation. In a second series of experiments the gap junction inhibitor, 18alpha-glycyrrhetinic acid (GA), was utilised. An initial experiment determined that GA dose-dependently blocked OCC metabolic coupling (0.2% coupling at 10 microM compared with 13.6% in controls). When HX-arrested CEO and DO were cultured for 17-18 h in medium containing increasing concentrations of GA, meiotic maturation was induced in CEO but not DO, suggesting that the cumulus cells provided a

  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. Regulation of gap junction channels by infectious agents and inflammation in the CNS

    PubMed Central

    Castellano, Paul; Eugenin, Eliseo A.

    2014-01-01

    Gap junctions (GJs) are conglomerates of intercellular channels that connect the cytoplasm of two or more cells, and facilitate the transfer of ions and small molecules, including second messengers, resulting in metabolic and electrical coordination. In general, loss of gap junctional communication (GJC) has been associated with cellular damage and inflammation resulting in compromise of physiological functions. Recently, it has become evident that GJ channels also play a critical role in the pathogenesis of infectious diseases and associated inflammation. Several pathogens use the transfer of intracellular signals through GJ channels to spread infection and toxic signals that amplify inflammation to neighboring cells. Thus, identification of the mechanisms by which several infectious agents alter GJC could result in new potential therapeutic approaches to reduce inflammation and their pathogenesis. PMID:24847208

  3. Effect of sound on gap-junction-based intercellular signaling: Calcium waves under acoustic irradiation

    NASA Astrophysics Data System (ADS)

    Deymier, P. A.; Swinteck, N.; Runge, K.; Deymier-Black, A.; Hoying, J. B.

    2015-11-01

    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.

  4. De novo reestablishment of gap junctional intercellular communications during reprogramming to pluripotency and differentiation.

    PubMed

    Sharovskaya, Yulia Y; Philonenko, Elena S; Kiselev, Sergei L; Lagarkova, Maria A

    2012-09-20

    Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ESCs) and various somatic cells. GJIC has been implicated in the regulation of cell proliferation, self-renewal, and differentiation. Recently, a new type of pluripotent stem cells was generated by direct reprogramming of somatic cells. Here, for the first time, we show that during reprogramming events GJIC is re-established upon reaching complete reprogramming. The opposite process of cell differentiation from the pluripotent state leads to the disruption of GJIC between pluripotent and differentiated cell subsets. However, GJIC is subsequently re-established de novo within each differentiated cell type in vitro, forming communication compartments within a histotype. Our results provide the important evidence that reestablisment of functional gap junctions to the level similar to human ESCs is an additional physiological characteristic of somatic cell reprogramming to the pluripotent state and differentiation to the specific cell type.

  5. Molecular cloning of cDNA for rat liver gap junction protein

    PubMed Central

    1986-01-01

    An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA. PMID:3013898

  6. Spontaneous calcium signals induced by gap junctions in a network model of astrocytes

    NASA Astrophysics Data System (ADS)

    Kazantsev, V. B.

    2009-01-01

    The dynamics of a network model of astrocytes coupled by gap junctions is investigated. Calcium dynamics of the single cell is described by the biophysical model comprising the set of three nonlinear differential equations. Intercellular dynamics is provided by the diffusion of inositol 1,4,5-trisphosphate (IP3) through gap junctions between neighboring astrocytes. It is found that the diffusion induces the appearance of spontaneous activity patterns in the network. Stability of the network steady state is analyzed. It is proved that the increase of the diffusion coefficient above a certain critical value yields the generation of low-amplitude subthreshold oscillatory signals in a certain frequency range. It is shown that such spontaneous oscillations can facilitate calcium pulse generation and provide a certain time scale in astrocyte signaling.

  7. Reversible inhibition of gap junctional communication by tamoxifen in cultured cardiac myocytes.

    PubMed

    Verrecchia, F; Hervé, J

    1997-05-01

    Gap junction channels provide a cell-to-cell conduction pathway for direct exchange of ions and small molecules. The intercellular diffusion of a fluorescent dye, quantified in cardiac myocytes from neonatal rats by monitoring the fluorescence recovery after photobleaching, was found to be interrupted after short-term exposure (15 min) to tamoxifen, an anti-oestrogen drug often used in the treatment of human breast cancer. This diffusional uncoupling was dose dependent, occurred in the concentration range 3-25 microM and reversed after tamoxifen withdrawal. Some possible mechanisms of junctional channel closure have been examined. The cytosolic calcium concentration, examined using the fluorescent indicator Indo-1, did not vary during the short-term action of tamoxifen. A second anti-oestrogen agent (clomiphene) was able to impair gap junctional communication, whereas a third (nafoxidine) had no effect. Protein-kinase-C-inhibitor properties of tamoxifen did not seem to be involved in its uncoupling action. The characteristics of tamoxifen's action (i.e. channel inhibition delay, active concentration range, reversibility, etc.) were very similar to the previously observed effects of several other lipophilic compounds (e. g. 17beta-oestradiol, etc.) on junctional channels, and to recently reported effects of tamoxifen on voltage-gated calcium currents.

  8. Horizontal cell gap junctions: single-channel conductance and modulation by dopamine.

    PubMed Central

    McHahon, D G; Knapp, A G; Dowling, J E

    1989-01-01

    Horizontal cells form an electrically coupled network for the transmission of inhibitory signals in the outer retina. In teleosts, horizontal cell coupling is modulated by the neurotransmitter dopamine. Using voltage-clamped pairs of teleost horizontal cells, we have examined the effects of dopamine on the conductance and gating properties of the cell-to-cell channels that mediate electrical synaptic transmission. Variance analysis of the junctional current noise showed that dopamine substantially reduced the open probability of gap junction channels, from 0.75 to 0.14. Direct observation of unitary junctional gating events in poorly coupled cell pairs indicated that these channels have a unitary conductance of 50-60 pS. The elementary conductance of channels in cell pairs treated with dopamine (48.7 +/- 6.6 pS) was statistically indistinguishable from channels in untreated cells (53.2 +/- 7.2 pS). Uncoupling with octanol also yielded a similar unitary conductance (61.1 +/- 11.1 pS). Our results suggest that dopamine reduces the open probability of gap junctional channels by decreasing their open duration. Images PMID:2477845

  9. Closing the proximity gap in a metallic Josephson junction between three superconductors

    NASA Astrophysics Data System (ADS)

    Padurariu, C.; Jonckheere, T.; Rech, J.; Mélin, R.; Feinberg, D.; Martin, T.; Nazarov, Yu. V.

    2015-11-01

    We describe the proximity effect in a short disordered metallic junction between three superconducting leads. Andreev bound states in the multiterminal junction may cross the Fermi level. We reveal that for a quasicontinuous metallic density of states, crossings at the Fermi level manifest as a closing of the proximity-induced gap. We calculate the local density of states for a wide range of transport parameters using quantum circuit theory. The gap closes inside an area of the space spanned by the superconducting phase differences. We derive an approximate analytic expression for the boundary of the area and compare it to the full numerical solution. The size of the area increases with the transparency of the junction and is sensitive to asymmetry. The finite density of states at zero energy is unaffected by the electron-hole decoherence present in the junction, although decoherence is important at higher energies. Our predictions can be tested using tunneling transport spectroscopy. To encourage experiments, we calculate the current-voltage characteristic in a typical measurement setup. We show how the structure of the local density of states can be mapped out from the measurement.

  10. 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

  11. Cell-based delivery of dATP via gap junctions enhances cardiac contractility.

    PubMed

    Lundy, Scott D; Murphy, Sean A; Dupras, Sarah K; Dai, Jin; Murry, Charles E; Laflamme, Michael A; Regnier, Michael

    2014-07-01

    The transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is a promising strategy to treat myocardial infarction and reverse heart failure, but to date the contractile benefit in most studies remains modest. We have previously shown that the nucleotide 2-deoxyadenosine triphosphate (dATP) can substitute for ATP as the energy substrate for cardiac myosin, and increasing cellular dATP content by globally overexpressing ribonucleotide reductase (R1R2) can dramatically enhance cardiac contractility. Because dATP is a small molecule, we hypothesized that it would diffuse readily between cells via gap junctions and enhance the contractility of neighboring coupled wild type cells. To test this hypothesis, we performed studies with the goals of (1) validating gap junction-mediated dATP transfer in vitro and (2) investigating the use of R1R2-overexpressing hPSC-CMs in vivo as a novel strategy to increase cardiac function. We first performed intracellular dye transfer studies using dATP conjugated to fluorescein and demonstrated rapid gap junction-mediated transfer between cardiomyocytes. We then cocultured wild type cardiomyocytes with either cardiomyocytes or fibroblasts overexpressing R1R2 and saw more than a twofold increase in the extent and rate of contraction of wild type cardiomyocytes. Finally, we transplanted hPSC-CMs overexpressing R1R2 into healthy uninjured rat hearts and noted an increase in fractional shortening from 41±4% to 53±5% just five days after cell transplantation. These findings demonstrate that dATP is an inotropic factor that spreads between cells via gap junctions. Our data suggest that transplantation of dATP-producing hPSC-CMs could significantly increase the effectiveness of cardiac cell therapy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Pharmacological blockade of gap junctions induces repetitive surging of extracellular potassium within the locust CNS.

    PubMed

    Spong, Kristin E; Robertson, R Meldrum

    2013-10-01

    The maintenance of cellular ion homeostasis is crucial for optimal neural function and thus it is of great importance to understand its regulation. Glial cells are extensively coupled by gap junctions forming a network that is suggested to serve as a spatial buffer for potassium (K(+)) ions. We have investigated the role of glial spatial buffering in the regulation of extracellular K(+) concentration ([K(+)]o) within the locust metathoracic ganglion by pharmacologically inhibiting gap junctions. Using K(+)-sensitive microelectrodes, we measured [K(+)]o near the ventilatory neuropile while simultaneously recording the ventilatory rhythm as a model of neural circuit function. We found that blockade of gap junctions with either carbenoxolone (CBX), 18β-glycyrrhetinic acid (18β-GA) or meclofenamic acid (MFA) reliably induced repetitive [K(+)]o surges and caused a progressive impairment in the ability to maintain baseline [K(+)]o levels throughout the treatment period. We also show that a low dose of CBX that did not induce surging activity increased the vulnerability of locust neural tissue to spreading depression (SD) induced by Na(+)/K(+)-ATPase inhibition with ouabain. CBX pre-treatment increased the number of SD events induced by ouabain and hindered the recovery of [K(+)]o back to baseline levels between events. Our results suggest that glial spatial buffering through gap junctions plays an essential role in the regulation of [K(+)]o under normal conditions and also contributes to a component of [K(+)]o clearance following physiologically elevated levels of [K(+)]o. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. 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.

  14. The Role of Chemical Inhibition of Gap-Junctional Intercellular Communication in Toxicology

    DTIC Science & Technology

    1991-03-31

    Florida. Inhibition of gap junctional intercellular communication (GJIC has been implicated as an important epigenetic modulation during...Annual Meeting of the Society of Toxicology, February 1991, Dallas, Texas. A major epigenetic modulation induced by many tumor promoters both in vivo...GJIC in rat pancreatic epithelial cells. The results indicated that many chlorinated pesticides , the phorbol ester tumor promoter, TPA, and a number of

  15. Mathematical modeling of gap junction coupling and electrical activity in human β-cells

    NASA Astrophysics Data System (ADS)

    Loppini, Alessandro; Braun, Matthias; Filippi, Simonetta; Gram Pedersen, Morten

    2015-12-01

    Coordinated insulin secretion is controlled by electrical coupling of pancreatic β-cells due to connexin-36 gap junctions. Gap junction coupling not only synchronizes the heterogeneous β-cell population, but can also modify the electrical behavior of the cells. These phenomena have been widely studied with mathematical models based on data from mouse β-cells. However, it is now known that human β-cell electrophysiology shows important differences to its rodent counterpart, and although human pancreatic islets express connexin-36 and show evidence of β-cell coupling, these aspects have been little investigated in human β-cells. Here we investigate theoretically, the gap junction coupling strength required for synchronizing electrical activity in a small cluster of cells simulated with a recent mathematical model of human β-cell electrophysiology. We find a lower limit for the coupling strength of approximately 20 pS (i.e., normalized to cell size, ˜2 pS pF-1) below which spiking electrical activity is asynchronous. To confront this theoretical lower bound with data, we use our model to estimate from an experimental patch clamp recording that the coupling strength is approximately 100-200 pS (10-20 pS pF-1), similar to previous estimates in mouse β-cells. We then investigate the role of gap junction coupling in synchronizing and modifying other forms of electrical activity in human β-cell clusters. We find that electrical coupling can prolong the period of rapid bursting electrical activity, and synchronize metabolically driven slow bursting, in particular when the metabolic oscillators are in phase. Our results show that realistic coupling conductances are sufficient to promote synchrony in small clusters of human β-cells as observed experimentally, and provide motivation for further detailed studies of electrical coupling in human pancreatic islets.

  16. Extracellular Space Attenuates the Effect of Gap Junctional Remodeling on Wave Propagation: A Computational Study

    PubMed Central

    Cabo, Candido; Boyden, Penelope A.

    2009-01-01

    Abstract Ionic channels and gap junctions are remodeled in cells from the 5-day epicardial border zone (EBZ) of the healing canine infarct. The main objective of the study was to determine the effect of gap junctional conductance (Gj) remodeling and Cx43 redistribution to the lateral membrane on conduction velocity (θ) and anisotropic ratio, and how gap junctional remodeling is modulated by the extracellular space. We first implemented subcellular monodomain and two-domain computer models of normal epicardium (NZ) to understand how extracellular space modulates the relationship between Gj and θ in NZ. We found that the extracellular space flattens the Gj-θ relationship, thus θ becomes less sensitive to changes in Gj. We then investigated the functional consequences of Gj remodeling and Cx43 distribution in subcellular computer models of cells of the outer pathway (IZo) and central pathway (IZc) of reentrant circuits. In IZo cells, side-to-side (transverse) Gj is 10% the value in NZ cells. Such Gj remodeling causes a 45% decrease in transverse θ (θT). Inclusion of an extracellular space reduces the decrease in θT to 31%. In IZc cells, Cx43 redistribution along the lateral membrane results in a 29% increase in θT. That increase in θT is a consequence of the decrease in access resistance to the Cx43 plaques that occur with the Cx43 redistribution. Extracellular space reduces the increase in θT to 10%. In conclusion: 1), The extracellular space included in normal epicardial simulations flattens the Gj-θ relationship with θ becoming less sensitive to changes in Gj. 2), The extracellular space attenuates the effects of gap junction epicardial border zone remodeling (i.e., Gj reduction and Cx43 lateralization) on θT. PMID:19383455

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

    PubMed Central

    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. PMID:26771544

  18. The Role of Chemical Inhibition of Gap Junctional Intercellular Communication in Toxicology.

    DTIC Science & Technology

    1988-02-14

    cell communication, tumor promoters, terato- 0620 gens, neurotoxins, protein kinase C, chemical toxicity. 19 ABSTRACT (Continue on reverse if necessary...hypothesis that chemical modulation of gap junctional intercellular communication can lead to many toxic endpoints, such as teratogenesis, tumor promotion... tumor promotion, reproductive-, immune- and neurotoxicities. To date, after two years into the project, we have initiated work on all of the specific aims

  19. Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa.

    PubMed

    Le Bourhis, Mikaël; Rimbaud, Stéphanie; Grebert, Denise; Congar, Patrice; Meunier, Nicolas

    2014-09-01

    Several factors modulate the first step of odour detection in the rat olfactory mucosa (OM). Among others, vasoactive peptides such as endothelin might play multifaceted roles in the different OM cells. Like their counterparts in the central nervous system, the olfactory sensory neurons are encompassed by different glial-like non-neuronal OM cells; sustentacular cells (SCs) surround their cell bodies, whereas olfactory ensheathing cells (OECs) wrap their axons. Whereas SCs maintain both the structural and ionic integrity of the OM, OECs assure protection, local blood flow control and guiding of olfactory sensory neuron axons toward the olfactory bulb. We previously showed that these non-neuronal OM cells are particularly responsive to endothelin in vitro. Here, we confirmed that the endothelin system is strongly expressed in the OM using in situ hybridization. We then further explored the effects of endothelin on SCs and OECs using electrophysiological recordings and calcium imaging approaches on both in vitro and ex vivo OM preparations. Endothelin induced both robust calcium signals and gap junction uncoupling in both types of cells. This latter effect was mimicked by carbenoxolone, a known gap junction uncoupling agent. However, although endothelin is known for its antiapoptotic effect in the OM, the uncoupling of gap junctions by carbenoxolone was not sufficient to limit the cellular death induced by serum deprivation in OM primary culture. The functional consequence of the endothelin 1-induced reduction of the gap junctional communication between OM non-neuronal cells thus remains to be elucidated. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  20. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

  1. [Gap junctional intercellular communication: a new mechanism in pathophysiology of migraine with aura. Therapeutic applications].

    PubMed

    Sarrouilhe, D; Dejean, C

    2012-12-01

    Migraine is a common, recurrent and disabling primary headache disorder, which affects up to 20% of the population. About a third of patients with migraine have attacks with aura, a focal neurological disturbance that manifests itself as visual, sensitive or motor symptoms. Cortical spreading depression, a wave of electrical activity that moves across the cerebral cortex through neuronal-glial cell gap junctions, would be involved in the triggering of migraine aura. Moreover, cortical spreading depression activates perivascular trigeminal afferents in the neocortex, that through central and peripheral reflex, cause inflammatory reaction in the meninges to generate the headache. Tonabersat, a novel benzopyran compound, was selected for clinical trial on the basis of its inhibitory activity on cortical spreading depression and neurogenic inflammation in animal models of migraine. Moreover, tonabersat inhibited trigeminal ganglion neuronal-glial cell gap junctions, suggesting that this compound could prevent peripheral sensitization within the ganglion. In clinical trial, tonabersat showed a preventive effect on attacks of migraine with aura but had no efficacy on non-aura attacks and in the acute treatment of migraine. In conclusion, neuronal-glial cell gap junctional intercellular communication seems to be involved in the pathophysiology of migraine with aura and is emerging as a new promising therapeutic target for prophylactic treatment of patients with chronic attacks.

  2. 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.

  3. 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

  4. Gap junctions linking the dendritic network of GABAergic interneurons in the hippocampus.

    PubMed

    Fukuda, T; Kosaka, T

    2000-02-15

    The network of GABAergic interneurons connected by chemical synapses is a candidate for the generator of synchronized oscillations in the hippocampus. We present evidence that parvalbumin (PV)-containing GABAergic neurons in the rat hippocampal CA1 region, known to form a network by mutual synaptic contacts, also form another network connected by dendrodendritic gap junctions. Distal dendrites of PV neurons run parallel to the alveus (hippocampal white matter) and establish multiple contacts with one another at the border between the stratum oriens and the alveus. In electron microscopic serial section analysis, gap junctions could be identified clearly at 24% of these contact sites. A dendrodendritic chemical synapse and a mixed synapse also were found between PV-immunoreactive dendrites. Three-dimensional reconstruction of the dendritic arborization revealed that both PV neurons of the well known vertical type (presumptive basket cells and axoaxonic cells) and those of another horizontal type constitute the dendritic network at the light microscopic level. The extent of dendritic fields of single PV neurons in the lateral direction was 538 +/- 201 micrometer (n = 5) in the vertical type and 838 +/- 159 micrometer (n = 6) in the horizontal type. Our previous and present observations indicate that PV-containing GABAergic neurons in the hippocampus form the dual networks connected by chemical and electrical synapses located at axosomatic and dendrodendritic contact sites, respectively. Gap junctions linking the dendritic network may mediate coherent synaptic inputs to distant interneurons and thereby facilitate the synchronization of oscillatory activities generated in the interneuron network.

  5. 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.

  6. Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by beta-amyloid.

    PubMed

    Même, William; Calvo, Charles-Félix; Froger, Nicolas; Ezan, Pascal; Amigou, Edwige; Koulakoff, Annette; Giaume, Christian

    2006-03-01

    Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)-astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were identified as being the main factors responsible for this conditioned medium-mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimer's disease, at sites of beta-amyloid (Abeta) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL-1beta and TNF-alpha activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

  7. Phosphorylation of Connexin 50 by Protein Kinase A Enhances Gap Junction and Hemichannel Function*

    PubMed Central

    Liu, Jialu; Ek Vitorin, Jose F.; Weintraub, Susan T.; Gu, Sumin; Shi, Qian; Burt, Janis M.; Jiang, Jean X.

    2011-01-01

    Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers. PMID:21454606

  8. Assembly of the cochlear gap junction macromolecular complex requires connexin 26.

    PubMed

    Kamiya, Kazusaku; Yum, Sabrina W; Kurebayashi, Nagomi; Muraki, Miho; Ogawa, Kana; Karasawa, Keiko; Miwa, Asuka; Guo, Xueshui; Gotoh, Satoru; Sugitani, Yoshinobu; Yamanaka, Hitomi; Ito-Kawashima, Shioko; Iizuka, Takashi; Sakurai, Takashi; Noda, Tetsuo; Minowa, Osamu; Ikeda, Katsuhisa

    2014-04-01

    Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

  9. Experimental Platform for Studying Thermoelectric Properties in Vacuum Gaps and Molecular Junctions

    NASA Astrophysics Data System (ADS)

    Jeong, Wonho; Kim, Youngsang; Kim, Kyeongtae; Lee, Woochul; Reddy, Pramod

    2014-03-01

    Electromigrated break junction (EBJ) based molecular devices have enabled many research groups to study nanoscale charge transport. Although EBJ devices have been extensively used due to the advantages of a three terminal configuration in tuning the electronic structure, it has not been possible to use them to study thermoelectric properties. This is because creating temperature differentials across the nanogap of EBJs is technically challenging. In order to overcome this experimental limitation, we carefully designed and created a new experimental platform (EBJIH, EBJ with integrated heater) that enables us to study thermoelectric properties in vacuum gaps and molecular junctions. To prove that temperature differentials can be established in these three terminal devices, we performed nanometer resolution thermal imaging using scanning thermal microscopy under UHV conditions. The results clearly show that temperature differentials can indeed be established in the devices. Further, we have used these devices to study the thermoelectric properties of vacuum gaps between gold electrodes and found that the thermoelectric properties were very sensitive to gap dimensions. We are also currently adopting this platform to study thermoelectric properties in molecular junctions.

  10. Physiologic regulation of heart rate and blood pressure involves connexin 36–containing gap junctions

    PubMed Central

    Lall, Varinder K.; Bruce, Gareth; Voytenko, Larysa; Drinkhill, Mark; Wellershaus, Kerstin; Willecke, Klaus; Deuchars, Jim; Deuchars, Susan A.

    2017-01-01

    Chronically elevated sympathetic nervous activity underlies many cardiovascular diseases. Elucidating the mechanisms contributing to sympathetic nervous system output may reveal new avenues of treatment. The contribution of the gap junctional protein connexin 36 (Cx36) to the regulation of sympathetic activity and thus blood pressure and heart rate was determined using a mouse with specific genetic deletion of Cx36. Ablation of the Cx36 protein was confirmed in sympathetic preganglionic neurons of Cx36-knockout (KO) mice. Telemetric analysis from conscious Cx36 KO mice revealed higher variance in heart rate and blood pressure during rest and activity compared to wild-type (WT) mice, and smaller responses to chemoreceptor activation when anesthetized. In the working heart–brain stem preparation of the Cx36-KO mouse, respiratory-coupled sympathetic nerve discharge was attenuated and responses to chemoreceptor stimulation and noxious stimulation were blunted compared to WT mice. Using whole cell patch recordings, sympathetic preganglionic neurons in spinal cord slices of Cx36-KO mice displayed lower levels of spikelet activity compared to WT mice, indicating reduced gap junction coupling between neurons. Cx36 deletion therefore disrupts normal regulation of sympathetic outflow with effects on cardiovascular parameters.—Lall, V. K., Bruce, G., Voytenko, L., Drinkhill, M., Wellershaus, K., Willecke, K., Deuchars, J., Deuchars, S. A. Physiologic regulation of heart rate and blood pressure involves connexin 36–containing gap junctions. PMID:28533325

  11. Slow conduction and gap junction remodeling in murine ventricle after chronic alcohol ingestion

    PubMed Central

    2011-01-01

    Background Long-term heavy alcohol drinkers are prone to the development of cardiac arrhythmia. To understand the mechanisms, we evaluated the cardiac structural and electrophysiological changes in mice chronically drinking excessive alcohol. Results Male C57BL/6J mice were given 36% alcohol in the drinking water. Those given blank water were used as control. Twelve weeks later, the phenotypic characteristics of the heart, including gap junctions and electrical properties were examined. In the alcohol group the ventricles contained a smaller size of cardiomyocytes and a higher density of capillary networks, compared to the control. Western blots showed that, after drinking alcohol, the content of connexin43 (Cx43) protein in the left ventricle was increased by 18% (p < 0.05). Consistently, immunoconfocal microscopy demonstrated that Cx43 gap junctions were up-regulated in the alcohol group with a disorganized distribution, compared to the control. Optical mapping showed that the alcohol group had a reduced conduction velocity (40 ± 18 vs 60 ± 7 cm/sec, p < 0.05) and a higher incidence of ventricular tachyarrhythmia (62% vs 30%, p < 0.05). Conclusion Long-term excessive alcohol intake resulted in extensive cardiac remodeling, including changes in expression and distribution of gap junctions, growth of capillary network, reduction of cardiomyocyte size, and decrease of myocardial conduction. PMID:21955691

  12. Activity-Dependent Neuronal Control of Gap-Junctional Communication in Astrocytes

    PubMed Central

    Rouach, Nathalie; Glowinski, Jacques; Giaume, Christian

    2000-01-01

    A typical feature of astrocytes is their high degree of intercellular communication through gap junction channels. Using different models of astrocyte cultures and astrocyte/neuron cocultures, we have demonstrated that neurons upregulate gap-junctional communication and the expression of connexin 43 (Cx43) in astrocytes. The propagation of intercellular calcium waves triggered in astrocytes by mechanical stimulation was also increased in cocultures. This facilitation depends on the age and number of neurons, indicating that the state of neuronal differentiation and neuron density constitute two crucial factors of this interaction. The effects of neurons on astrocytic communication and Cx43 expression were reversed completely after neurotoxic treatments. Moreover, the neuronal facilitation of glial coupling was suppressed, without change in Cx43 expression, after prolonged pharmacological treatments that prevented spontaneous synaptic activity. Altogether, these results demonstrate that neurons exert multiple and differential controls on astrocytic gap-junctional communication. Since astrocytes have been shown to facilitate synaptic efficacy, our findings suggest that neuronal and astrocytic networks interact actively through mutual setting of their respective modes of communication. PMID:10871289

  13. Sequence and developmental expression of mRNA coding for a gap junction protein in Xenopus

    PubMed Central

    1988-01-01

    Cloned complementary DNAs representing the complete coding sequence for an embryonic gap junction protein in the frog Xenopus laevis have been isolated and sequenced. The cDNAs hybridize with an RNA of 1.5 kb that is first detected in gastrulating embryos and accumulates throughout gastrulation and neurulation. By the tailbud stage, the highest abundance of the transcript is found in the region containing ventroposterior endoderm and the rudiment of the liver. In the adult, transcripts are present in the lungs, alimentary tract organs, and kidneys, but are not detected in the brain, heart, body wall and skeletal muscles, spleen, or ovary. The gene encoding this embryonic gap junction protein is present in only one or a few copies in the frog genome. In vitro translation of RNA synthesized from the cDNA template produces a 30-kD protein, as predicted by the coding sequence. This product has extensive sequence similarity to mammalian gap junction proteins in its putative transmembrane and extracellular domains, but has diverged substantially in two of its intracellular domains. PMID:2843548

  14. Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

    SciTech Connect

    Iwase, Yumiko . E-mail: Iwase.Yumiko@mg.m-pharma.co.jp; Fukata, Hideki . E-mail: fukata@faculty.chiba-u.jp; Mori, Chisato . E-mail: cmori@faculty.chiba-u.jp

    2006-05-01

    Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17{beta}-estradiol (E{sub 2}, a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 {mu}M DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 {mu}M E{sub 2} and 25 {mu}M GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at {mu}M level between DES and E{sub 2} on GJIC inhibition was observed, but not between GEN and E{sub 2}. DES and E{sub 2} showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 {mu}M was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E{sub 2} (10 pM and 20 {mu}M) and GEN (25 {mu}M) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E{sub 2} and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual nongenotoxic mechanism including PKC pathway on testicular carcinogenesis or development.

  15. Calcium-induced calcium release and gap junctions mediate large-scale calcium waves in olfactory ensheathing cells in situ.

    PubMed

    Stavermann, Maren; Meuth, Patrick; Doengi, Michael; Thyssen, Anne; Deitmer, Joachim W; Lohr, Christian

    2015-08-01

    Olfactory ensheathing cells (OECs) are a specialised type of glial cells, supporting axon growth and guidance during development and regeneration of the olfactory nerve and the nerve layer of the olfactory bulb. We measured calcium signalling in OECs in olfactory bulb in-toto preparations using confocal and epifluorescence microscopy and the calcium indicator Fluo-4. We identified two subpopulations of olfactory bulb OECs: OECs in the outer sublamina of the nerve layer responded to purinergic neurotransmitters such as adenosine triphosphate with calcium transients, while OECs in the inner sublamina of the nerve layer did not respond to neurotransmitters. However, the latter generated spontaneous calcium waves that covered hundreds of cells. These calcium waves persisted in the presence of tetrodotoxin and in calcium-free saline, but were abolished after calcium store depletion with cyclopiazonic acid or inositol trisphosphate receptor blockage with 2-APB. Calcium waves could be triggered by laser photolysis of caged inositol trisphosphate. Blocking purinoceptors with PPADS had no effect on calcium wave propagation, whereas blocking gap junctions with carbenoxolone or meclofenamic acid entirely suppressed calcium waves. Increasing calcium buffer capacity in OECs with NP-EGTA ("caged" Ca(2+)) prevented calcium wave generation, and laser photolysis of NP-EGTA in a small group of OECs resulted in a calcium increase in the irradiated cells followed by a calcium wave. We conclude that calcium waves in OECs can be initiated by calcium-induced calcium release via InsP3 receptors and propagate through gap junctions, while purinergic signalling is not involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress.

    PubMed

    Zhang, Zongqi; Chen, Yizhu; Zhang, Tiantian; Guo, Lingyu; Yang, Wenlong; Zhang, Junfeng; Wang, Changqian

    2016-01-01

    Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm2 or 5 dyn/cm2. The myoendothelial gap junctions were evaluated by using a multi-photon microscope. In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm2 increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm2 promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. The exposure of HCAECs to shear stress of 5 dyn/cm2 caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis. © 2016 The Author(s) Published by S. Karger AG

  17. Mild hypothermia preserves myocardial conduction during ischemia by maintaining gap junction intracellular communication and Na(+) channel function.

    PubMed

    Nassal, Michelle M J; Wan, Xiaoping; Dale, Zack; Deschênes, Isabelle; Wilson, Lance D; Piktel, Joseph S

    2017-05-01

    Acute cardiac ischemia induces conduction velocity (CV) slowing and conduction block, promoting reentrant arrhythmias leading to sudden cardiac arrest. Previously, we found that mild hypothermia (MH; 32°C) attenuates ischemia-induced conduction block and CV slowing in a canine model of early global ischemia. Acute ischemia impairs cellular excitability and the gap junction (GJ) protein connexin (Cx)43. We hypothesized that MH prevented ischemia-induced conduction block and CV slowing by preserving GJ expression and localization. Canine left ventricular preparations at control (36°C) or MH (32°C) were subjected to no-flow prolonged (30 min) ischemia. Optical action potentials were recorded from the transmural left ventricular wall, and CV was measured throughout ischemia. Cx43 and Na(+) channel (NaCh) remodeling was assessed using both confocal immunofluorescence (IF) and/or Western blot analysis. Cellular excitability was determined by microelectrode recordings of action potential upstroke velocity (dV/dtmax) and resting membrane potential (RMP). NaCh current was measured in isolated canine myocytes at 36 and 32°C. As expected, MH prevented conduction block and mitigated ischemia-induced CV slowing during 30 min of ischemia. MH maintained Cx43 at the intercalated disk (ID) and attenuated ischemia-induced Cx43 degradation by both IF and Western blot analysis. MH also preserved dV/dtmax and NaCh function without affecting RMP. No difference in NaCh expression was seen at the ID by IF or Western blot analysis. In conclusion, MH preserves myocardial conduction during prolonged ischemia by maintaining Cx43 expression at the ID and maintaining NaCh function. Hypothermic preservation of GJ coupling and NaCh may be novel antiarrhythmic strategies during resuscitation.NEW & NOTEWORTHY Therapeutic hypothermia is now a class I recommendation for resuscitation from cardiac arrest. This study determined that hypothermia preserves gap junction coupling as well as Na

  18. Modulation of human cell responses to space radiation by gap-junction communication

    NASA Astrophysics Data System (ADS)

    Autsavapromporn, Narongchai; de Toledo, Sonia M.; Buonanno, Manuela; Yang, Zhi; Harris, Andrew; Jay-Gerin, Jean-Paul; Azzam, Edouard

    Understanding the biological effects of space radiation and their underlying mechanism is critical to estimating the health risk associated with human exploration of space. A coordinated interaction of multiple cellular processes is likely involved in the sensing and processing of stressful effects induced by different types of space radiation. Here, we focused on the role of gap-junction intercellular communication (GJIC) in responses of human cells exposed to 1 GeV/n protons or 56 Fe-ions. We compared the results with data obtained in human cells exposed, in parallel, to γ-rays or α-particles. As expected, a higher level of cell killing and DNA damage, per unit dose, was induced in confluent, density-inhibited cells (98% in G0 /G1 ) exposed to α-particles or energetic 56 Fe-ions than γ-rays or protons. Strikingly, greatly attenuated effects occurred when sub-confluent cultures, synchronized in G0 /G1 ,were exposed to 56 Fe-ions. These data suggest that direct intercellular communication is involved in the effects of high linear energy transfer (LET) 56 Fe-ions. To examine the role of gap-junctions in propagating stressful effect, confluent cultures were exposed to 56 Fe-ions or α-particles and incubated for various time periods at 37° C in the presence or absence of the gap-junction inhibitor α-glycyrrhetinic acid (AGA). No repair of potentially lethal radiation damage occurred in cells incubated in the absence of AGA. In contrast, inhibition of functional GJIC significantly enhanced clonogenic survival of irradiated cells. To test the role of junctional channel permeability in the observed effects, we used human adenocarcinoma (HeLa) cells in which specific connexins (Cx) can be expressed in the absence of endogenous connexins. Whereas HeLa cells with selective inducible expression of Cx26 gap-junctions promoted radiation toxic effects, expression of Cx32 junctional channels in HeLa cells promoted pro-survival effects. Experiments are in progress to

  19. Akinesia and freezing caused by Na(+) leak-current channel (NALCN) deficiency corrected by pharmacological inhibition of K(+) channels and gap junctions.

    PubMed

    Kasap, Merve; Bonnett, Kendra; Aamodt, Eric J; Dwyer, Donard S

    2017-04-01

    The Na(+) leak-current channel (NALCN) regulates locomotion, respiration, and intellectual development. Previous work highlighted striking similarities between characteristic movement phenotypes of NALCN-deficient animals (Drosophila and Caenorhabditis elegans) and the major symptoms of Parkinson's disease and primary progressive freezing gait. We have discovered novel physiological connections between the NALCN, K(+) channels, and gap junctions that mediate regulation of locomotion in C. elegans. Drugs that block K(+) channels and gap junctions or that activate Ca(++) channels significantly improve movement of NALCN-deficient animals. Loss-of-function of the NALCN creates an imbalance in ions, including K(+) and Ca(++) , that interferes with normal cycles of depolarization-repolarization. This work suggests new therapeutic strategies for certain human movement disorders. J. Comp. Neurol. 525:1109-1121, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Intraglomerular gap junctions enhance interglomerular synchrony in a sparsely connected olfactory bulb network.

    PubMed

    Pouille, Frederic; McTavish, Thomas S; Hunter, Lawrence E; Restrepo, Diego; Schoppa, Nathan E

    2017-09-01

    Despite sparse connectivity, population-level interactions between mitral cells (MCs) and granule cells (GCs) can generate synchronized oscillations in the rodent olfactory bulb. Intraglomerular gap junctions between MCs at the same glomerulus can greatly enhance synchronized activity of MCs at different glomeruli. The facilitating effect of intraglomerular gap junctions on interglomerular synchrony is through triggering of mutually synchronizing interactions between MCs and GCs. Divergent connections between MCs and GCs make minimal direct contribution to synchronous activity. A dominant feature of the olfactory bulb response to odour is fast synchronized oscillations at beta (15-40 Hz) or gamma (40-90 Hz) frequencies, thought to be involved in integration of olfactory signals. Mechanistically, the bulb presents an interesting case study for understanding how beta/gamma oscillations arise. Fast oscillatory synchrony in the activity of output mitral cells (MCs) appears to result from interactions with GABAergic granule cells (GCs), yet the incidence of MC-GC connections is very low, around 4%. Here, we combined computational and experimental approaches to examine how oscillatory synchrony can nevertheless arise, focusing mainly on activity between 'non-sister' MCs affiliated with different glomeruli (interglomerular synchrony). In a sparsely connected model of MCs and GCs, we found first that interglomerular synchrony was generally quite low, but could be increased by a factor of 4 by physiological levels of gap junctional coupling between sister MCs at the same glomerulus. This effect was due to enhanced mutually synchronizing interactions between MC and GC populations. The potent role of gap junctions was confirmed in patch-clamp recordings in bulb slices from wild-type and connexin 36-knockout (KO) mice. KO reduced both beta and gamma local field potential oscillations as well as synchrony of inhibitory signals in pairs of non-sister MCs. These effects were

  1. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions

    PubMed Central

    Valiunas, V; Polosina, YY; Miller, H; Potapova, IA; Valiuniene, L; Doronin, S; Mathias, RT; Robinson, RB; Rosen, MR; Cohen, IS; Brink, PR

    2005-01-01

    The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin-deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK-kcdc and Mβ16tsA-kcdc) were co-cultured with labelled wild type, NRK-wt or Mβ16tsA-wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT-PCR and immunoblotting. Co-culture of Mβ16tsA-kcdc cells with Mβ16tsA-wt, N2A or NRK-wt cells had no effect on pol β levels in these cells. Similarly, co-culture of NRK-kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co-culture of NRK-kcdc with NRK-wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA-kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell-to-cell movement of the siRNA. These results support the novel hypothesis

  2. Structural organization of the dendritic reticulum linked by gap junctions in layer 4 of the visual cortex.

    PubMed

    Fukuda, Takaichi

    2017-01-06

    Neuronal gap junctions are ubiquitous in the brain, but their precise positions in actual neuronal circuits have been uncertain, and their functional roles remain unclear. In this study, I visualized connexin36-immunoreactive gap junctions and examined the structural features of the interconnected dendrites arising from parvalbumin (PV)-positive interneurons in layer 4 of the feline visual cortex. I observed evidence for net-like dense linkages of dendrites among virtually all PV neurons (56/58 cells, 96.6%) in the tissue. This dendritic reticulum established connections among clustered cells and further among remote cells. The latter connectivity exhibited a preference for vertical direction, including translaminar linkages, but was also characterized by lateral continuity. Measurement of the distances from each dendritic gap junction back to the two connected somata revealed that at least one of two somata was within 50μm from the junction in 77.5% of the cases and within 75μm in 91.2% of the cases. Thus, distal gap junctions mediated morphologically asymmetrical connection where one soma was close to, but the other soma was far from the connecting junction. This connectivity was typically observed between neurons located apart in the same columnar space, where a long vertical dendrite bridged two neurons through an asymmetrically positioned gap junction. In contrast, gap junctions formed between nearby cells were close to both somata. Thalamocortical afferents established synapses densely on somata of layer 4 PV neurons, indicating the possible involvement of proximal gap junctions in visual stimulus-driven feedforward regulation. These findings provide a new structural basis for cortical investigations. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  3. Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

    PubMed Central

    Jin, Chengshi; Martyn, Kendra D.; Kurata, Wendy E.; Warn-Cramer, Bonnie J.; Lau, Alan F.

    2010-01-01

    Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions −3 and −2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43ΔI382) or the last five amino acids (Cx43Δ378–382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43ΔI382 and Cx43Δ378–382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation. PMID:16247852

  4. Combinational treatment of gap junctional activator and tamoxifen in breast cancer cells.

    PubMed

    Gakhar, Gunjan; Hua, Duy H; Nguyen, Thu Annelise

    2010-01-01

    Tamoxifen is a drug of choice for endocrine-responsive breast tumor patients. However, tamoxifen resistance has become a major concern for the treatment of breast cancer. Combinational therapies of tamoxifen and different drugs are being frequently studied. In this study, we tested the efficacy of substituted quinolines (code name=PQ1; a gap junctional activator) in combination with tamoxifen in T47D cells. Colony growth assay was performed using soft agar to measure the colony growth, whereas cell proliferation was measured by the MTT assay in T47D cells. The level of Ki67, survivin, and BAX was measured using confocal microscopy along with western blot analysis. Apoptosis-bromodeoxyuridine triphosphate labeling was also examined in the induced treatment of T47D cells. We observed a 55% decrease in the colony growth in the presence of combination of PQ1 and tamoxifen, whereas tamoxifen alone had little effect. A combination of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1 resulted in only 16% cell viability compared with controls at 48 h in T47D cells by the MTT assay. We found a significant increase in BAX protein at 1 h in the presence of 500 nmol/l PQ1 alone, 10 micromol/l tamoxifen alone, and the combination of PQ1 and tamoxifen. A two-fold increase was observed in active caspase 3 in the presence of combinational treatment of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1. In addition, flow cytometric analysis showed a 50% increase in the number of apoptotic cells in the presence of the combination of tamoxifen and PQ1 compared with the control. Furthermore, the results show that combinational treatment of tamoxifen and PQ1 significantly reduces the expression of survivin in T47D cells. Gap junction inhibitor studies with carbenexolone were also performed confirming the role of gap junctions in cell proliferation and cell death. The combinational treatment of PQ1 and tamoxifen has a significant increase in BAX expression, caspase 3 activation, and DNA

  5. Analysis of Full-Test tools and their limitations as applied to terminal junction blocks

    NASA Technical Reports Server (NTRS)

    Smith, J. L.

    1983-01-01

    Discovery of unlocked contacts in Deutsch Block terminal junctions in Solid Rocket Booster flight hardware prompted an investigation into pull test techniques to help insure against possible failures. Internal frictional forces between socket and pin and between wire and grommet were examined. Pull test force must be greater than internal friction yet less than the crimp strength of the pin or socket. For this reason, a 100 percent accurate test is impossible. Test tools were evaluated. Available tools are adequate for pull testing.

  6. 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

  7. Degradation and resynthesis of gap junction protein in plasma membranes of regenerating liver after partial hepatectomy or cholestasis

    PubMed Central

    Traub, Otto; Drüge, Petra Maria; Willecke, Klaus

    1983-01-01

    Changes in the total amount of the gap junction protein (Mr 26,000) after partial hepatectomy or bile duct ligation and recanalization were investigated in rat liver membranes by quantitative immunoblot with rabbit antiserum to the Mr 26,000 protein. The loss and reappearance of the Mr 26,000 protein roughly paralleled loss and reappearance of gap junction plaques analyzed previously under similar physiological conditions by freeze-fracture of hepatocyte surfaces. The total amount of the hepatic Mr 26,000 protein in liver plasma membranes and the total area of the hepatocyte surface occupied by gap junction plaques appeared to be proportional under these conditions. However, at the minimum, 28-35 hr after partial hepatectomy we still find about 15% of the Mr 26,000 protein, in contrast to <1% of gap junction plaques, determined by morphometric analysis. This discrepancy is probably due to the fact that very small gap junction plaques, single connexons, and free Mr 26,000 gap junction subunits are missed by the morphometric analysis. At the times of the minimal amount of the Mr 26,000 protein in hepatic plasma membranes after partial hepatectomy or bile duct ligation we found that crude hepatic lysosomal membranes of these rats contained less Mr 26,000 protein than lysosomal membranes of nonoperated control animals. Thus, we conclude that the decrease and increase of the total amount of the Mr 26,000 protein cannot be explained only by dispersal and reuse of gap junction subunits but are largely due to degradation and resynthesis of the Mr 26,000 protein. No significant change in the amount of the Mr 21,000 protein that had been isolated with gap junction plaques was observed in liver plasma membranes after partial hepatectomy. This confirms our previous conclusion that the Mr 26,000 and Mr 21,000 proteins are independent of each other. Images PMID:6298773

  8. 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.

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

    PubMed Central

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

    2011-01-01

    Abstract Cell–cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca2+]i) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca2+]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. KATP 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 [Ca2+]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. [Ca2+]i was further elevated under basal conditions, but insulin release still suppressed in KATP 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 [Ca2+]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. PMID:21930600

  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 in the Teleost Fish Lineage: Duplicated Connexins May Contribute to Skin Pattern Formation and Body Shape Determination

    PubMed Central

    Watanabe, Masakatsu

    2017-01-01

    Gap junctions are intercellular channels that allow passage of ions and small molecules between adjacent cells. Gap junctions in vertebrates are composed of connexons, which are an assembly of six proteins, connexins. Docking of two connexons on the opposite cell surfaces forms a gap junction between the cytoplasm of two neighboring cells. Connexins compose a family of structurally related four-pass transmembrane proteins. In mammals, there are ~20 connexins, each of which contributes to unique permeability of gap junctions, and mutations of some connexin-encoding genes are associated with human diseases. Zebrafish has been predicted to contain 39 connexin-encoding genes; the high number can be attributed to gene duplication during fish evolution, which resulted in diversified functions of gap junctions in teleosts. The determination of body shapes and skin patterns in animal species is an intriguing question. Mathematical models suggest principle mechanisms explaining the diversification of animal morphology. Recent studies have revealed the involvement of gap junctions in fish morphological diversity, including skin pattern formation and body shape determination. This review focuses on connexins in teleosts, which are integrated in the mathematical models explaining morphological diversity of animal skin patterns and body shapes. PMID:28271062

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

    PubMed Central

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

    2016-01-01

    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 Ca2+ 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. PMID:27051341

  13. Ginsenoside Rg1-induced antidepressant effects involve the protection of astrocyte gap junctions within the prefrontal cortex.

    PubMed

    Jin, Can; Wang, Zhen-Zhen; Zhou, Heng; Lou, Yu-Xia; Chen, Jiao; Zuo, Wei; Tian, Man-Tong; Wang, Zhi-Qi; Du, Guo-Hua; Kawahata, Ichiro; Yamakuni, Tohru; Zhang, Yi; Chen, Nai-Hong; Zhang, Dan-Shen

    2017-04-03

    Ginsenoside Rg1 (Rg1) exhibits antidepressant-like activity by increasing neurogenesis and dendritic spine density without discernible side effects. However, the molecular mechanisms underlying Rg1 antidepressant activity remain poorly understood. As the dysfunction of gap junctions between astrocytes in the prefrontal cortex (PFC) is implicated in major depression disorder, the aim of this study was to investigate the effects of Rg1 on astrocyte gap junctions in the PFC. Rats exposed to chronic unpredictable stress (CUS) were administered Rg1 (5, 10, and 20mg/kg) for 28days and analyzed for depressive symptoms using the sucrose preference and forced swimming tests. Functional and morphological changes of gap junction channels in the PFC were evaluated using dye transfer and electron microscopy, respectively. The expression of connexin 43 (Cx43) was analyzed by western blotting. Rg1 markedly alleviated depression-like behavior in rats. Long-term Rg1 treatment of CUS-exposed rats also significantly prevented the decrease in dye diffusion and improved the ultrastructure of astrocyte gap junctions in the PFC, indicating beneficial effects on the functional activity of gap junction channels in the brain. In addition, Rg1 upregulated Cx43 expression in the PFC reduced by CUS exposure, which significantly correlated with its antidepressant-like effects. The results demonstrate that Rg1-induced antidepressant effects are might be mediated, in part, by protecting astrocyte gap junctions within the prefrontal cortex.

  14. Age-related changes in gap junctional intercellular communication in osteoblastic cells

    PubMed Central

    Genetos, Damian C.; Zhou, Zhiyi; Li, Zhongyong; Donahue, Henry J.

    2013-01-01

    Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions—membrane-spanning channels that allow direct cell-to-cell conductance of small signaling molecules—are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated GJIC. PTH demonstrated age-dependent effects upon GJIC: osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC. PMID:22696456

  15. Cell-Cell Interaction Proteins (Gap Junctions, Tight Junctions, and Desmosomes) and Water Transporter Aquaporin 4 in Meningothelial Cells of the Human Optic Nerve.

    PubMed

    Zeleny, Thi Ngoc Co; Kohler, Corina; Neutzner, Albert; Killer, Hanspeter E; Meyer, Peter

    2017-01-01

    Meningothelial cells (MECs) play a central role in the maintenance of cerebrospinal fluid (CSF) homeostasis and in physiological and pathophysiological processes within the subarachnoid space (SAS) linking them to optic nerve (ON) pathologies. Still, not much is known about their structural properties that might enable MECs to perform specific functions within the ON microenvironment. For closer characterization of the structural properties of the human MEC layer in the arachnoid, we performed immunohistological analyses to evaluate the presence of cell-cell interaction markers, namely, markers for tight junctions (JAM1, Occludin, and Claudin 5), gap junctions (Connexin 26 and 43), and desmosomes (Desmoplakin) as well as for water channel marker aquaporin 4 (AQP4) in retrobulbar, midorbital, and intracanalicular human ON sections. MECs displayed immunopositivity for markers of tight junctions (JAM1, Occludin, and Claudin 5) and gap junctions (Connexin 26 and 43) as well as for AQP4 water channels. However, no immunopositivity was found for Desmoplakin. MECs are connected via tight junctions and gap junctions, and they possess AQP4 water channels. The presence of these proteins emphasizes the important function of MECs within the ON microenvironment as part of the meningeal barrier. Beyond this barrier function, the expression of these proteins by MECs supports a broader role of these cells in signal transduction and CSF clearance pathways within the ON microenvironment.

  16. Endogenous protein phosphatase 1 runs down gap junctional communication of rat ventricular myocytes.

    PubMed

    Duthe, F; Plaisance, I; Sarrouilhe, D; Hervé, J C

    2001-11-01

    Gap junctional channels are essential for normal cardiac impulse propagation. In ventricular myocytes of newborn rats, channel opening requires the presence of ATP to allow protein kinase activities; otherwise, channels are rapidly deactivated by the action of endogenous protein phosphatases (PPs). The lack of influence of Mg(2+) and of selective PP2B inhibition is not in favor of the involvements of Mg(2+)-dependent PP2C and PP2B, respectively, in the loss of channel activity. Okadaic acid (1 microM) and calyculin A (100 nM), both inhibitors of PP1 and PP2A activities, significantly retarded the loss of channel activity. However, a better preservation was obtained in the presence of selective PP1 inhibitors heparin (100 microg/ml) or protein phosphatase inhibitor 2 (I2; 100 nM). Conversely, the stimulation of endogenous PP1 activity by p-nitrophenyl phosphate, in the presence of ATP, led to a progressive fading of junctional currents unless I2 was simultaneously added. Together, these results suggest that a basal phosphorylation-dephosphorylation turnover regulates gap junctional communication which is rapidly deactivated by PP1 activity when the phosphorylation pathway is hindered.

  17. Properties of gap junction channels formed by Cx46 alone and in combination with Cx50.

    PubMed Central

    Hopperstad, M G; Srinivas, M; Spray, D C

    2000-01-01

    Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties. PMID:11023900

  18. Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

    PubMed

    Yazaki, I; Dale, B; Tosti, E

    1999-08-15

    Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction. Copyright 1999 Academic Press.

  19. Fluxon dynamics in two-gap superconductor-based long Josephson junction

    NASA Astrophysics Data System (ADS)

    Ghimire, Bal Ram

    A superconducting tunnel junction with two-gap superconductors, such as MgB2 and iron-based superconductors, can lead to more interesting phase dynamics than those with one-gap superconductors. The phase dynamics in a long Josephson junction (LJJ) may be described by using the sine-Gordon equation. The difference in the phase dynamics between the LJJ with two-gap superconductors and that with the one-gap superconductors arises due to the presence of multiple tunneling channels between the superconductor (S) layers and the inter-band Josephson effect within the same S layer. The inter-band Josephson effect leads to both spatial and temporal modulation of the critical current between the two adjacent S layers. In this work, the effects of critical current modulation on the trajectories of the single Josephson vortex (i.e., fluxon) and the current-voltage characteristics of the two-gap superconductor-based LJJ are estimated. Also, the possibility of a broken time-reversal symmetry state ground state of a single LJJ due to the presence of additional tunneling channels is investigated by using a microscopic model for two-gap superconductors. The consequence of this broken time reversal ground state is discussed. Finally, the equation of motion for fluxon for coupled LJJs interacting via both the magnetic induction effect and charging effect is investigated. As the inter-band Josephson effect is found to affect the dynamics of a single fluxon in a single LJJ, this effect is explicitly taken into account for a two-coupled LJJ stack. This equation of motion is expected to be an excellent starting point for exploring interesting LJJ properties such as collective dynamics of fluxons as well as fractional fluxons.

  20. Retinoids regulate the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

    PubMed

    Kelsey, Linda; Katoch, Parul; Johnson, Kristen E; Batra, Surinder K; Mehta, Parmender P

    2012-01-01

    The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.

  1. Band-gaps in long Josephson junctions with periodic phase-shifts

    NASA Astrophysics Data System (ADS)

    Ahmad, Saeed; Susanto, Hadi; Wattis, Jonathan A. D.

    2017-04-01

    We investigate analytically and numerically a long Josephson junction on an infinite domain, having arbitrary periodic phase shift of κ, that is, the so-called 0-κ long Josephson junction. The system is described by a one-dimensional sine-Gordon equation and has relatively recently been proposed as artificial atom lattices. We discuss the existence of periodic solutions of the system and investigate their stability both in the absence and presence of an applied bias current. We find critical values of the phase-discontinuity and the applied bias current beyond which static periodic solutions cease to exist. Due to the periodic discontinuity in the phase, the system admits regions of allowed and forbidden bands. We perturbatively investigate the Arnold tongues that separate the region of allowed and forbidden bands, and discuss the effect of an applied bias current on the band-gap structure. We present numerical simulations to support our analytical results.

  2. 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.

  3. 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.

  4. Association of temporary complete AV block and junctional ectopic tachycardia after surgery for congenital heart disease.

    PubMed

    Paech, Christian; Dähnert, Ingo; Kostelka, Martin; Mende, Meinhardt; Gebauer, Roman

    2015-01-01

    Junctional ectopic tachycardia (JET) is a postoperative complication with a mortality rate of up to 14% after surgery for congenital heart disease. This study evaluated the risk factors of JET and explored the association of postoperative temporary third degree atrioventricular (AV) block and the occurrence of JET. Data were collected retrospectively from 1158 patients who underwent surgery for congenital heart disease. The overall incidence of JET was 2.8%. Temporary third degree AV block occurred in 1.6% of cases. Permanent third degree AV block requiring pacemaker implantation occurred in 1% of cases. In all, 56% of patients with JET had temporary AV block (P < 0.001), whereas no case of postoperative JET was reported in patients with permanent AV block (P = 0.56). temporary third degree AV block did not suffer from JET. A correlation between temporary third degree AV block and postoperative JET could be observed. The risk factors identified for JET include younger age groups at the time of surgery, longer aortic cross clamping time and surgical procedures in proximity to the AV node.

  5. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    SciTech Connect

    Zou, Hui; Zhuo, Liling; Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong; Liu, Zongping

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  6. 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.

  7. Connexin50 couples axon terminals of mouse horizontal cells by homotypic gap junctions.

    PubMed

    Dorgau, Birthe; Herrling, Regina; Schultz, Konrad; Greb, Helena; Segelken, Jasmin; Ströh, Sebastian; Bolte, Petra; Weiler, Reto; Dedek, Karin; Janssen-Bienhold, Ulrike

    2015-10-01

    Horizontal cells in the mouse retina are of the axon-bearing B-type and contribute to the gain control of photoreceptors and to the center-surround organization of bipolar cells by providing feedback and feedforward signals to photoreceptors and bipolar cells, respectively. Horizontal cells form two independent networks, coupled by dendro-dendritic and axo-axonal gap junctions composed of connexin57 (Cx57). In Cx57-deficient mice, occasionally the residual tracer coupling of horizontal cell somata was observed. Also, negative feedback from horizontal cells to photoreceptors, potentially mediated by connexin hemichannels, appeared unaffected. These results point to the expression of a second connexin in mouse horizontal cells. We investigated the expression of Cx50, which was recently identified in axonless A-type horizontal cells of the rabbit retina. In the mouse retina, Cx50-immunoreactive puncta were predominantly localized on large axon terminals of horizontal cells. Electron microscopy did not reveal any Cx50-immunolabeling at the membrane of horizontal cell tips invaginating photoreceptor terminals, ruling out the involvement of Cx50 in negative feedback. Moreover, Cx50 colocalized only rarely with Cx57 on horizontal cell processes, indicating that both connexins form homotypic rather than heterotypic or heteromeric gap junctions. To check whether the expression of Cx50 is changed when Cx57 is lacking, we compared the Cx50 expression in wildtype and Cx57-deficient mice. However, Cx50 expression was unaffected in Cx57-deficient mice. In summary, our results indicate that horizontal cell axon terminals form two independent sets of homotypic gap junctions, a feature which might be important for light adaptation in the retina. © 2015 Wiley Periodicals, Inc.

  8. 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

  9. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    SciTech Connect

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    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 the 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.

  10. 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

  11. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    PubMed

    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

  12. Gap junctions in dorsal root ganglia: possible contribution to visceral pain.

    PubMed

    Huang, Tian-Ying; Belzer, Vitali; Hanani, Menachem

    2010-01-01

    Peripheral injuries can lead to sensitization of neurons in dorsal root ganglia (DRGs), which can contribute to chronic pain. The neurons are sensitized by a combination of physiological and biochemical changes, whose full details are still obscure. Another cellular element in DRGs are satellite glial cells (SGCs), which surround the neurons, but little is known about their role in nociception. We investigated the contribution of SGCs to neuronal sensitization in isolated S1 DRGs from a mouse model of colonic inflammation induced by local application of dinitrosulfonate benzoate (DNBS). Retrograde labeling was used to identify DRG neurons projecting to the colon. Cell-to-cell coupling was determined by intracellular dye injection, and the electrical properties of the neurons were studied with intracellular electrodes. Pain behavior was assessed with von-Frey hairs. The dye injections showed that 10-12 days after DNBS application there was a 6.6-fold increase in gap junction-mediated coupling between SGCs surrounding adjacent neurons, and this occurred preferentially (another 2-fold increase) near neurons that project to the colon. Neuron-neuron coupling incidence increased from 0.7% to 12.1% by colonic inflammation. Inflammation led to an augmented neuronal excitability, and to a reduced pain threshold. Gap junction blockers abolished the inflammation-induced changes in SGCs and neurons, and significantly reversed the pain behavior. We propose that inflammation induces augmented cell coupling in DRGs that contributes to neuronal hyperexcitability, which in turn leads to visceral pain. Gap junction blockers may have potential as analgesic drugs.

  13. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    PubMed

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  14. Voltage-dependent gating of single gap junction channels in an insect cell line.

    PubMed Central

    Bukauskas, F F; Weingart, R

    1994-01-01

    De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely. PMID:7524710

  15. Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness.

    PubMed

    Kikuchi, T; Adams, J C; Miyabe, Y; So, E; Kobayashi, T

    2000-01-01

    In the mammalian cochlea, there are two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. Thus far, four different connexin molecules, including connexin 26, 30, 31, and 43, have been reported in the cochlea. The two networks of gap junctions form the route by which K+ ions that pass through the sensory cells during mechanosensory transduction can be recycled back to the endolymphatic space, from which they reenter the sensory cells. Activation of hair cells by acoustic stimuli induces influx of K+ ions from the endolymph to sensory hair cells. These K+ ions are released basolaterally to the extracellular space of the organ of Corti, from which they enter the cochlear supporting cells. Once inside the supporting cells they move via the epithelial cell gap junction system laterally to the lower part of the spiral ligament. The K+ ions are released into the extracellular space of the spiral ligament by root cells and taken up by type II fibrocytes. This uptake incorporates K+ into the connective tissue gap junction system. Within this system, the K+ ions pass through the tight junctional barrier of the stria vascularis and are released within the intrastrial extracellular space. The marginal cells of the stria vascularis then take up K+ and return it to the endolymphatic space, where it can be used again in sensory transduction. It is highly probable that mutations of connexin genes that result in human nonsyndromic deafness cause dysfunction of cochlear gap junctions and thereby interrupt K+ ion recirculation pathways. In addition to connexin mutations, other conditions may disrupt gap junctions within the ear. For example, mice with a functionally significant mutation of Brain-4, which is expressed in the connective tissue cells within the cochlea, show marked depression of the endolymphatic potential and profound sensorineural hearing loss. It seems likely that disruption of connective

  16. Peptides and peptide-derived molecules targeting the intracellular domains of Cx43: gap junctions versus hemichannels.

    PubMed

    Iyyathurai, Jegan; D'hondt, Catheleyne; Wang, Nan; De Bock, Marijke; Himpens, Bernard; Retamal, Mauricio A; Stehberg, Jimmy; Leybaert, Luc; Bultynck, Geert

    2013-12-01

    About a decade ago, the molecular determinants controlling the opening and closing of Cx43 gap junction channels have been identified. Advanced biophysical approaches revealed a critical role for structural rearrangements in the cytoplasmic loop and dimerization of the C-terminal tail, resulting in binding of the C-terminal tail to the cytoplasmic loop and Cx43 gap junction channel closure during cellular acidosis. This has spurred the development of Cx43-mimetic peptides and peptidomimetics that interfere with these loop/tail interactions, thereby preventing the closure of Cx43 gap junctions, e.g. in the heart upon ischemia. Recently, we found that loop/tail interactions control Cx43-hemichannel activity but with an opposite effect. Binding of the C-terminal tail to the cytoplasmic loop is a requisite for the opening of Cx43 hemichannels in response to different stimuli, like decreased extracellular [Ca2+], increased intracellular [Ca2+], positive membrane potentials or ischemia. Strikingly, peptides that favor the open state of Cx43 gap junctions like the L2 peptide inhibit Cx43-hemichannel opening. These tools now provide unprecedented opportunities to selectively inhibit Cx43 hemichannels while maintaining Cx43 gap junction communication, impossible to achieve with siRNA or knockdown approaches both affecting gap junctions and hemichannels. These tools not only are very helpful to unravel the role of Cx43 hemichannels in complex biological systems, but also hold therapeutic potential to counteract excessive Cx43-hemichannel activity like in ischemia/reperfusion in the brain and the heart or to prevent Cx43 hemichannel-mediated gliotransmitter release in the basal amygdala during memory consolidation in response to emotional events. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'. Copyright © 2013. Published by Elsevier Ltd.

  17. Gap Junctions Enhance the Antiproliferative Effect of MicroRNA-124-3p in Glioblastoma Cells.

    PubMed

    Suzhi, Zhang; Liang, Tao; Yuexia, Peng; Lucy, Liu; Xiaoting, Hong; Yuan, Zhang; Qin, Wang

    2015-10-01

    MicroRNA (miRNA) holds promise as a novel therapeutic tool for cancer treatment. However, the transfection efficiency of current delivery systems represents a bottleneck for clinical applications. Here, we demonstrate that gap junctions mediate an augmentative effect on the antiproliferation mediated by miR-124-3p in U87 and C6 glioblastoma cells. The functional inhibition of gap junctions using either siRNA or pharmacological inhibition eliminated the miR-124-3p-mediated antiproliferation, whereas the enhancement of gap junctions with retinoic acid treatment augmented this miR-124-3p-mediated antiproliferation. A similar effect was observed in glioblastoma xenograft models. More importantly, patch clamp and co-culture assays demonstrated the transmission of miR-124-3p through gap junction channels into adjacent cells. In further exploring the impact of gap junction-mediated transport of miR-124-3p on miR-124-3p target pathways, we found that miR-124-3p inhibited glioblastoma cell growth in part by decreasing the protein expression of cyclin-dependent kinase 6, leading to cell cycle arrest at the G0 /G1 phase; moreover, pharmacological regulation of gap junctions affected this cell cycle arrest. In conclusion, our results indicate that the "bystander" effects of functional gap junctions composed of connexin 43 enhance the antitumor effect of miR-124-3p in glioblastoma cells by transferring miR-124-3p to adjacent cells, thereby enhancing G0 /G1 cell cycle arrest. These observations provide a new guiding strategy for the clinical application of miRNA therapy in tumor treatment. © 2015 Wiley Periodicals, Inc.

  18. Ultrastructure of smooth muscle, gap junctions and glycogen distribution in Taenia solium tapeworms from experimentally infected hamsters.

    PubMed

    Willms, Kaethe; Robert, Lilia; Caro, José Antonio

    2003-03-01

    Taenia solium adults were grown in hamsters infected by feeding them with cysticerci from pig carcasses. Viable strobilae were collected from the hamster duodenum 20-60 days post-infection, fixed and processed for transmission electron microscopy (TEM). Fourteen strobilae were cut into pieces and embedded in individual blocks. Sections, stained with toluidine blue, were then photographed by light microscopy. Over 1,200 TEM images were obtained from selected blocks. Maturing proglottids exhibited a dense myofilament lattice of connecting fibers, each contained in sarcoplamsic extensions of myocytons and emitting cytoplasmic processes loosely attached to other cells, structures characterized as myocyton-myofilament-pseudopod units, which are interpreted as structures involved in the transport of cells and membrane-bound-glycogen from the germinative tissues to mature proglottids. Densely packed membrane-bound glycogen particles were found between the tegumentary cytons of the neck tissue, and as single-stranded particles between the tegumentary cytons of mature proglottids. These were wrapped around cell bodies in the parenchyma of maturing proglottids and as thin cytoplasmic strands between the testicular lobules of mature proglottids. A large number of cell-to-cell adhesions were identified as gap junctions connected to glycogen strands. We suggest that these are involved in the transport of glucose to differentiating tissues.

  19. Influence of the molecular structure of steroids on their ability to interrupt gap junctional communication.

    PubMed

    Hervé, J C; Pluciennik, F; Verrecchia, F; Bastide, B; Delage, B; Joffre, M; Délèze, J

    1996-02-01

    17 beta-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 microM, whereas its intracellular application at 1 microM totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism.

  20. Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.

    PubMed Central

    Suchyna, T M; Nitsche, J M; Chilton, M; Harris, A L; Veenstra, R D; Nicholson, B J

    1999-01-01

    The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that

  1. Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

    PubMed Central

    Wang, Hong-Zhan; Veenstra, Richard D.

    1997-01-01

    The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (γj, in pS) of the junctional current-voltage relationships in 115 mM XCl were RbCl (103) ≥ CsCl (102) > KCl (97) > NaCl (79) ≥ LiCl (78) > TMACl (65) > TEACl (53) and for 115 mM KY were KBr (105) > KCl (97) > Kacetate (77) > Kglutamate (61). The single channel conductance-aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KCl. Instead, the conductance of the rCx43 channel in 115 mM KCl was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor Dx/Do, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent cations and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 ± 0.4 Å. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore. PMID

  2. A Computational Approach to Detect Gap Junction Plaques and Associate Them with Cells in Fluorescent Images

    PubMed Central

    Goldberg, Joshua S.; Vadakkan, Tegy J.; Hirschi, Karen K.

    2013-01-01

    Intercellular signaling is a fundamental requirement for complex biological system function and survival. Communication between adjoining cells is largely achieved via gap junction channels made up of multiple subunits of connexin proteins, each with unique selectivity and regulatory properties. Intercellular communication via gap junction channels facilitates transmission of an array of cellular signals, including ions, macromolecules, and metabolites that coordinate physiological processes throughout tissues and entire organisms. Although current methods used to quantify connexin expression rely on number or area density measurements in a field of view, they lack cellular assignment, distance measurement capabilities (both within the cell and to extracellular structures), and complete automation. We devised an automated computational approach built on a contour expansion algorithm platform that allows connexin protein detection and assignment to specific cells within complex tissues. In addition, parallel implementation of the contour expansion algorithm allows for high-throughput analysis as the complexity of the biological sample increases. This method does not depend specifically on connexin identification and can be applied more widely to the analysis of numerous immunocytochemical markers as well as to identify particles within tissues such as nanoparticles, gene delivery vehicles, or even cellular fragments such as exosomes or microparticles. PMID:23324867

  3. Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face.

    PubMed

    McGonnell, I M; Green, C R; Tickle, C; Becker, D L

    2001-11-01

    Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face.

  4. Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro

    PubMed Central

    Eugenín, Eliseo A.; González, Hernán E.; Sánchez, Helmuth A.; Brañes, María C.; Sáez, Juan C.

    2007-01-01

    Connexin43 (Cx43), a gap junction protein subunit, has been previously detected in Kupffer cells (KCs) during liver inflammation, however, KCs phagocytose cell debris that may include Cx43 protein, which could explain the detection of Cx43 in KCs. We determined that KCs express Cx43 and form gap junctions both in vivo and in vitro. In liver sections of animals treated with LPS, Cx43 was detected at ED2+ cells interfaces, indicating formation of GJ between KCs in vivo. In vitro, unstimulated KCs cultures did not form functional GJs, and expressed low levels of Cx43 that showed a diffuse intracellular distribution. In contrast, KCs treated with LPS plus IFN-γ, expressed a greater amount of Cx43 at both the, protein and mRNA levels, and showed Cx43 at cell-cell contacts associated with higher dye coupling. In conclusion, activation of KCs in vivo or in vitro resulted in enhanced Cx43 expression levels and formation of GJ that might play relevant roles during liver inflammation. PMID:17900549

  5. Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance.

    PubMed

    Sakurai, Takashi; Tsuchida, Mariko; Lampe, Paul D; Murakami, Masahiro

    2013-08-15

    Cardiac remodeling resulting from impairment of myocardial integrity leads to heart failure, through still incompletely understood mechanisms. The fibroblast growth factor (FGF) system has been implicated in tissue maintenance, but its role in the adult heart is not well defined. We hypothesized that the FGF system plays a role in the maintenance of cardiac homeostasis, and the impairment of cardiomyocyte FGF signaling leads to pathological cardiac remodeling. We showed that FGF signaling is required for connexin 43 (Cx43) localization at cell-cell contacts in isolated cardiomyocytes and COS7 cells. Lack of FGF signaling led to decreased Cx43 phosphorylation at serines 325/328/330 (S325/328/330), sites known to be important for assembly of gap junctions. Cx43 instability induced by FGF inhibition was restored by the Cx43 S325/328/330 phospho-mimetic mutant, suggesting FGF-dependent phosphorylation of these sites. Consistent with these in vitro findings, cardiomyocyte-specific inhibition of FGF signaling in adult mice demonstrated mislocalization of Cx43 at intercalated discs, whereas localization of N-cadherin and desmoplakin was not affected. This led to premature death resulting from impaired cardiac remodeling. We conclude that cardiomyocyte FGF signaling is essential for cardiomyocyte homeostasis through phosphorylation of Cx43 at S325/328/330 residues which are important for the maintenance of gap junction.

  6. Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

    PubMed Central

    Reed, K E; Westphale, E M; Larson, D M; Wang, H Z; Veenstra, R D; Beyer, E C

    1993-01-01

    Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins. Images PMID:7680674

  7. Gap junction channel. Its aqueous nature as indicated by deuterium oxide effects

    SciTech Connect

    Verselis, V.; Brink, P.R.

    1986-11-01

    The effects of temperature and solvent substitution with deuterium oxide (D2O) on axoplasmic (ga) and gap junctional (gj) conductances were examined in the earthworm septate median giant axon (MGA). The temperature coefficients (Q10) for ga and gj were 1.4 and 1.5, respectively, between 5 and 15 degrees C. Substitution with D/sub 2/O rapidly reduced both ga and gj by 20% and increased the Q10's to 1.5 and 1.8, respectively. The reduction in ga upon substitution with D/sub 2/O and with cooling in either solvent reflects the changes that occur in solvent viscosity, which indicates that ion mobility in axoplasm, as in free solution, is primarily governed by viscous properties of the solvent. The similar initial reduction observed for gj suggests that solvent occupies the gap junction channel volume and influences transjunctional ion mobility. With time there was a further reduction in gj at 20 degrees C and a larger Q10 in D/sub 2/O. The enhanced effects of D/sub 2/O on gj cannot be accounted for by solvent viscosity alone and may be due to an increased hydration of the channels and/or the transport ions and by isotope effects of hydrogen-deuterium exchange on the channel protein that reduce gj.

  8. Gap Junctions Are Essential for Generating the Correlated Spike Activity of Neighboring Retinal Ganglion Cells

    PubMed Central

    Paul, David L.; Wang, Jack T.; Huberman, Andrew D.; Bloomfield, Stewart A.

    2013-01-01

    Neurons throughout the brain show spike activity that is temporally correlated to that expressed by their neighbors, yet the generating mechanism(s) remains unclear. In the retina, ganglion cells (GCs) show robust, concerted spiking that shapes the information transmitted to central targets. Here we report the synaptic circuits responsible for generating the different types of concerted spiking of GC neighbors in the mouse retina. The most precise concerted spiking was generated by reciprocal electrical coupling of GC neighbors via gap junctions, whereas indirect electrical coupling to a common cohort of amacrine cells generated the correlated activity with medium precision. In contrast, the correlated spiking with the lowest temporal precision was produced by shared synaptic inputs carrying photoreceptor noise. Overall, our results demonstrate that different synaptic circuits generate the discrete types of GC correlated activity. Moreover, our findings expand our understanding of the roles of gap junctions in the retina, showing that they are essential for generating all forms of concerted GC activity transmitted to central brain targets. PMID:23936012

  9. Computational simulations of asymmetric fluxes of large molecules through gap junction channel pores.

    PubMed

    Mondal, Abhijit; Appadurai, Daniel A; Akoum, Nazem W; Sachse, Frank B; Moreno, Alonso P

    2017-01-07

    Gap junction channels are formed out of connexin isoforms, which enable molecule and ion selective diffusion amongst neighboring cells. HeLa cells expressing distinct connexins (Cx) allow the formation of heterotypic channels, where we observed a molecular charge-independent preferential flux of large fluorescent molecules in the Cx45 to Cx43 direction. We hypothesize that the pore's shape is a significant factor along-side charge and transjunctional voltages for this asymmetric flux. To test this hypothesis, we developed a 3D computational model simulating Brownian diffusion of large molecules in a gap junction channel pore. The basic pore contour was derived from x-ray crystallographic structures of Cx43 and Cx26 and approximated using basic geometric shapes. Lucifer yellow dye molecules and cesium counter-ions were modeled as spheres using their respective Stokes radii. Our simulation results from simple diffusion and constant concentration gradient experiments showed that only charged particles yield asymmetric fluxes in heterotypic pores. While increasing the inner mouth size resulted in a near-quadratic rise in flux, the rise was asymptotic for outer mouth radii increase. Probability maps and average force per particle per pore section explain the asymmetric flux with variation in pore shape. Furthermore, the simulation results are in agreement with our in vitro experimental results with HeLa cells in Cx43-Cx45 heterotypic configurations. The presence of asymmetric fluxes can help us to understand effects of the molecular structure of the pore and predict potential differences in vivo.

  10. The gap junctional protein INX-14 functions in oocyte precursors to promote C. elegans sperm guidance

    PubMed Central

    Edmonds, Johnathan W.; McKinney, Shauna L.; Prasain, Jeevan K.; Miller, Michael A.

    2011-01-01

    Innexins are the subunits of invertebrate gap junctions. Here we show that the innexin INX-14 promotes sperm guidance to the fertilization site in the C. elegans hermaphrodite reproductive tract. inx-14 loss causes cell nonautonomous defects in sperm migration velocity and directional velocity. Results from genetic and immunocytochemical analyses provide strong evidence that INX-14 acts in transcriptionally active oocyte precursors in the distal gonad, not in transcriptionally inactive oocytes that synthesize prostaglandin sperm-attracting cues. Somatic gonadal sheath cell interaction is necessary for INX-14 function, likely via INX-8 and INX-9 expressed in sheath cells. However, electron microscopy has not identified gap junctions in oocyte precursors, suggesting that INX-14 acts in a channel-independent manner or INX-14 channels are difficult to document. INX-14 promotes prostaglandin signaling to sperm at a step after F-series prostaglandin synthesis in oocytes. Taken together, our results support the model that INX-14 functions in a somatic gonad/germ cell signaling mechanism essential for sperm function. We propose that this mechanism regulates the transcription of a factor(s) that modulates prostaglandin metabolism, transport, or activity in the reproductive tract. PMID:21889935

  11. Phenobarbital specifically reduces gap junction protein mRNA level in rat liver.

    PubMed

    Mesnil, M; Fitzgerald, D J; Yamasaki, H

    1988-01-01

    The gene expression of liver major gap junction (GJ) protein was studied in rats systemically administered phenobarbital, a rat liver tumor promoter. Using a GJ protein cDNA and northern blot analysis, the level of GJ protein mRNA in liver was observed to be markedly reduced at 4 and 11 wk of phenobarbital exposure (0.1% in drinking water). However, the level of GJ protein mRNA was not altered in kidney at 11 wk of exposure. In liver, phenobarbital did not induce expression of the neoplasm-associated marker genes glutathione S-transferase (placental form) and gamma-glutamyltranspeptidase, while in kidney the observed expression of these genes was not changed. These in vivo results indicate that phenobarbital reduces GJ protein gene expression specifically in rat liver without altering expression of genes often altered during liver carcinogenesis, and they support assigning a role for the impairment of gap junctional intercellular communication in phenobarbital-mediated liver tumor promotion.

  12. Changes in gap junction protein (connexin 32) gene expression during rat liver carcinogenesis.

    PubMed

    Fitzgerald, D J; Mesnil, M; Oyamada, M; Tsuda, H; Ito, N; Yamasaki, H

    1989-10-01

    A rat liver gap junction (GJ) cDNA probe that detects mRNA encoding the 32 Kd GJ-protein (connexin 32) was employed to study GJ-protein gene expression in rat liver tumors induced by a single exposure to diethylnitrosamine (DEN) followed by exposure to 2-acetylaminofluorene (AAF)/CCl4/AAF or induced by systemic administration of N-ethyl-N-hydroxyethylnitrosamine (EHEN). All carcinomas generated by these carcinogens showed markedly reduced levels of GJ-protein mRNA. This may indicate that GJ-protein levels and gap-junctional intercellular communication (GJIC) capacity are also severely compromised. Moreover, all hyperplastic nodules also showed a reduced level of GJ-protein mRNA. Taken together with our earlier finding that the liver tumor promoter phenobarbital inhibits GJ-protein gene expression, these results suggest that deranged GJIC is a relatively early event in liver multistage carcinogenesis. A range of other cDNA probes was also used to characterize gene expression in the DEN-induced tumors. Induction of expression was seen for glutathione S-transferase (placental form) (GST-P), gamma-glutamyltranspeptidase (GGT), and c-raf but not for c-Ha-ras or c-myc.

  13. Th1 cells downregulate connexin 43 gap junctions in astrocytes via microglial activation

    PubMed Central

    Watanabe, Mitsuru; Masaki, Katsuhisa; Yamasaki, Ryo; Kawanokuchi, Jun; Takeuchi, Hideyuki; Matsushita, Takuya; Suzumura, Akio; Kira, Jun-ichi

    2016-01-01

    We previously reported early and extensive loss of astrocytic connexin 43 (Cx43) in acute demyelinating lesions of multiple sclerosis (MS) patients. Because it is widely accepted that autoimmune T cells initiate MS lesions, we hypothesized that infiltrating T cells affect Cx43 expression in astrocytes, which contributes to MS lesion formation. Primary mixed glial cell cultures were prepared from newborn mouse brains, and microglia were isolated by anti-CD11b antibody-conjugated magnetic beads. Next, we prepared astrocyte-rich cultures and astrocyte/microglia-mixed cultures. Treatment of primary mixed glial cell cultures with interferon (IFN) γ, interleukin (IL)-4, or IL-17 showed that only IFNγ or IL-17 at high concentrations reduced Cx43 protein levels. Upon treatment of astrocyte-rich cultures and astrocyte/microglia-mixed cultures with IFNγ, Cx43 mRNA/protein levels and the function of gap junctions were reduced only in astrocyte/microglia-mixed cultures. IFNγ-treated microglia-conditioned media and IL-1β, which was markedly increased in IFNγ-treated microglia-conditioned media, reduced Cx43 protein levels in astrocyte-rich cultures. Finally, we confirmed that Th1 cell-conditioned medium decreased Cx43 protein levels in mixed glial cell cultures. These findings suggest that Th1 cell-derived IFNγ activates microglia to release IL-1β that reduces Cx43 gap junctions in astrocytes. Thus, Th1-dominant inflammatory states disrupt astrocytic intercellular communication and may exacerbate MS. PMID:27929069

  14. In Situ Bipolar Electroporation for Localized Cell Loading with Reporter Dyes and Investigating Gap Junctional Coupling

    PubMed Central

    De Vuyst, Elke; De Bock, Marijke; Decrock, Elke; Van Moorhem, Marijke; Naus, Christian; Mabilde, Cyriel; Leybaert, Luc

    2008-01-01

    Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication. PMID:17872956

  15. Gap junction blockers: a potential approach to attenuate morphine withdrawal symptoms.

    PubMed

    Moradi, Sabah; Charkhpour, Mohammad; Ghavimi, Hamed; Motahari, Rasoul; Ghaderi, Majid; Hassanzadeh, Kambiz

    2013-10-21

    The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat. Adult male Wistar rats (225 - 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 μg/10 μl/rat, icv) or MFQ (50, 100 and 200 μg/10 μl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes. Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly. Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.

  16. A computational approach to detect gap junction plaques and associate them with cells in fluorescent images.

    PubMed

    Goldberg, Joshua S; Vadakkan, Tegy J; Hirschi, Karen K; Dickinson, Mary E

    2013-04-01

    Intercellular signaling is a fundamental requirement for complex biological system function and survival. Communication between adjoining cells is largely achieved via gap junction channels made up of multiple subunits of connexin proteins, each with unique selectivity and regulatory properties. Intercellular communication via gap junction channels facilitates transmission of an array of cellular signals, including ions, macromolecules, and metabolites that coordinate physiological processes throughout tissues and entire organisms. Although current methods used to quantify connexin expression rely on number or area density measurements in a field of view, they lack cellular assignment, distance measurement capabilities (both within the cell and to extracellular structures), and complete automation. We devised an automated computational approach built on a contour expansion algorithm platform that allows connexin protein detection and assignment to specific cells within complex tissues. In addition, parallel implementation of the contour expansion algorithm allows for high-throughput analysis as the complexity of the biological sample increases. This method does not depend specifically on connexin identification and can be applied more widely to the analysis of numerous immunocytochemical markers as well as to identify particles within tissues such as nanoparticles, gene delivery vehicles, or even cellular fragments such as exosomes or microparticles.

  17. Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas.

    PubMed

    Akopian, Abram; Kumar, Sandeep; Ramakrishnan, Hariharasubramanian; Viswanathan, Suresh; Bloomfield, Stewart A

    2016-07-13

    We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol., 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Self-organized synchronous oscillations in a network of excitable cells coupled by gap junctions.

    PubMed

    Lewis, T J; Rinzel, J

    2000-11-01

    Recent evidence suggests that electrical coupling plays a role in generating oscillatory behaviour in networks of neurons; however, the underlying mechanisms have not been identified. Using a cellular automata model proposed by Traub et al (Traub R D, Schmitz D, Jefferys J G and Draguhn A 1999 High-frequency population oscillations are predicted to occur in hippocampal pyramidal neural networks interconnected by axo-axonal gap junctions Neuroscience 92 407-26), we describe a novel mechanism for self-organized oscillations in networks that have strong, sparse random electrical coupling via gap junctions. The network activity is generated by random spontaneous activity that is moulded into regular population oscillations by the propagation of activity through the network. We explain how this activity gives rise to particular dependences of mean oscillation frequency on network connectivity parameters and on the rate of spontaneous activity, and we derive analytical expressions to approximate the mean frequency and variance of the oscillations. In doing so, we provide insight into possible mechanisms for frequency control and modulation in networks of neurons.

  19. Fabrication of 30 nm inter-electrode gap co-planar tunnel junctions with buried electrodes

    NASA Astrophysics Data System (ADS)

    Itoua, S.; Joachim, C.; Rousset, B.; Fabre, N.

    1994-05-01

    Co-planar tunnel junctions with a gap length in the 30 nm range have been fabricated using a 20 keV scanning electron microscope and a Au-Pd lift-off. The junction electrodes are less than 200 nm in width and are buried in the SiO2 substrate. This makes the gap surface accessible for atomic force microscope characterization and for local modification. Des jonctions tunnels co-planaires avec une largeur de coupure inférieure à 30 nm ont été fabriquées en utilisant un masqueur électronique à 20 keV et un procédé de lift-off d'un alliage Au-Pd. Les électrodes de la jonction ont moins de 200 nm de largeur et sont enterrées à la surface de SiO2. La mesure de la topographie de la surface de la coupure avec un microscope à force atomique montre une rugosité de moins de 1 nm.

  20. Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

    PubMed Central

    Yahalom, A; Warmbrodt, R D; Laird, D W; Traub, O; Revel, J P; Willecke, K; Epel, B L

    1991-01-01

    Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins. PMID:1668654

  1. Disruption of gap junctions reduces biomarkers of decidualization and angiogenesis and increases inflammatory mediators in human endometrial stromal cell cultures.

    PubMed

    Yu, Jie; Wu, Juanjuan; Bagchi, Indrani C; Bagchi, Milan K; Sidell, Neil; Taylor, Robert N

    2011-09-15

    Uterine decidualization is critical to embryonic implantation and sustained pregnancy. To evaluate the role of gap junction intercellular communications and connexin (Cx) proteins in the morphological and biochemical differentiation of decidualized human endometrial stromal cell (ESC) cultures. Translational cell biological study. Academic medical center. Endometrial tissue was provided by five healthy reproductive age women on no hormonal medication, undergoing laparoscopy in the early proliferative phase of the menstrual cycle. Endometrial biopsy under general anesthesia, establishment and decidualization of ESC with 10 nM 17β-estradiol, 100 nM progesterone and 0.5 mM dibutyryl-cAMP (E/P/c), and manipulation of gap junctions in vitro via a combination of pharmacological or transgenic approaches. Decidualized ESC evaluated morphologically for epithelioid transformation, gap junctions by dye diffusion and Cx43, prolactin, VEGF and IL-6 expression by RT-PCR, Western and ELISA methods. Cx43 accumulation and functional gap junctions between decidualized ESC increase concomitantly with morphological differentiation following E/P/c treatment. Disruption of gap junctions using pharmacological inhibitors or Cx43 shRNA prevents morphological differentiation and inhibits prolactin and VEGF secretion. By contrast, IL-6 secretion from decidualized ESC is augmented by both approaches. The findings suggest that decidualized ESC function as a coordinated secretory organ to regulate embryonic implantation via intercellular cooperation mediated by gap junctions. When adjacent cells can communicate through these junctions, decidual prolactin and VEGF secretion appears to be optimized for vascular development of the placental bed. Conversely, when intercellular communications are disrupted, angiogenesis is impaired and an inflammatory state is induced. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. The shaking B gene in Drosophila regulates the number of gap junctions between photoreceptor terminals in the lamina.

    PubMed

    Shimohigashi, M; Meinertzhagen, I A

    1998-04-01

    The molecular structure of insect gap junctions differs from that in vertebrates, and in Drosophila is possibly encoded by the shaking B (= Passover) locus. shaking B2 is a null allele that acts in the nervous system. In the shakB2 mutant, one site of action are gap junctions between photoreceptor terminals in the cartridges of the lamina, beneath the compound eye, which we assayed from the number of close-apposition profiles in thin-section EM. The number of profiles in the Canton-S (C-S) wild type is about 0.5 per cartridge per section in distal and mid-lamina depths, and significantly less, about one quarter this value, closer to the brain, in the proximal lamina. In shakB2, there are fewer profiles, approximately one quarter the number of appositions in distal and mid-lamina depths as in C-S, and their number does not differ significantly from those at the proximal depth in either the mutant or wild type. Thus mutant action is associated with a reduced number of appositions at distal and mid-lamina depths. We propose that R1-R6 gap junctions are partitioned into at least two strata, proximal and distal, and that two populations of gap junctions exist, one extending throughout the lamina that does not require shakB, and a second at distal and mid-depth levels, which does. The number of gap junctions is reduced in mutant shakB2, and surviving appositions at distal and middle lamina depths possibly have wider clefts than in C-S. Gap junctions are reduced equally between all R1-R6 terminals, so the two different types of junction proposed, shakB2- and non-shakB2-dependent, can apparently express in a single receptor terminal.

  3. 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.

  4. Dual Effects of Bilirubin on the Proliferation of Rat Renal NRK52E Cells and ITS Association with Gap Junctions

    PubMed Central

    Wang, Yanling; Zhu, Qiongfang; Luo, Chenfang; Zhang, Ailan; Hei, Ziqing; Su, Guangjie; Xia, Zhengyuan; Irwin, Michael G.

    2013-01-01

    Objective: The effect of bilirubin on renal pathophysiology is controversial. This study aimed to observe the effects of bilirubin on the proliferation of normal rat renal tubular epithelial cell line (NRK52E) and its potential interplay with gap junction function. Methods: Cultured NRK52E cells, seeded respectively at high- or low- densities, were treated with varying concentrations of bilirubin for 24 hours. Cell injury was assessed by measuring cell viability and proliferation, and gap junction function was assessed by Parachute dye-coupling assay. Connexin 43 protein was assessed by Western blotting. Results: At doses from 17.1 to 513μmol/L, bilirubin dose-dependently enhanced cell viability and colony-formation rates when cells were seeded at either high- or low- densities (all p<0.05 vs. solvent group) accompanied with enhanced intercellular fluorescence transmission and increased Cx43 protein expression in high-density cells. However, the above effects of BR were gradually reversed when its concentration increased from 684 to 1026μmol/L. In high-density cells, gap junction inhibitor 12-O-tetradecanoylphorbol 13-acetate attenuated bilirubin-induced enhancement of colony-formation and fluorescence transmission. However, in the presence of high concentration bilirubin (1026μmol/L), activation of gap junction with retinoid acid decreased colony-formation rates. Conclusion: Bilirubin can confer biphasic effects on renal NRK52E cell proliferation potentially by differentially affecting gap junction functions. PMID:23930103

  5. 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

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

    PubMed Central

    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

    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. PMID:26966678

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

    PubMed

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

    2014-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

    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.

  10. The assembly of developing motor neurons depends on an interplay between spontaneous activity, type II cadherins and gap junctions.

    PubMed

    Montague, Karli; Lowe, Andrew S; Uzquiano, Ana; Knüfer, Athene; Astick, Marc; Price, Stephen R; Guthrie, Sarah

    2017-03-01

    A core structural and functional motif of the vertebrate central nervous system is discrete clusters of neurons or 'nuclei'. Yet the developmental mechanisms underlying this fundamental mode of organisation are largely unknown. We have previously shown that the assembly of motor neurons into nuclei depends on cadherin-mediated adhesion. Here, we demonstrate that the emergence of mature topography among motor nuclei involves a novel interplay between spontaneous activity, cadherin expression and gap junction communication. We report that nuclei display spontaneous calcium transients, and that changes in the activity patterns coincide with the course of nucleogenesis. We also find that these activity patterns are disrupted by manipulating cadherin or gap junction expression. Furthermore, inhibition of activity disrupts nucleogenesis, suggesting that activity feeds back to maintain integrity among motor neurons within a nucleus. Our study suggests that a network of interactions between cadherins, gap junctions and spontaneous activity governs neuron assembly, presaging circuit formation. © 2017. Published by The Company of Biologists Ltd.

  11. A Gap Junction Protein, Inx2, Modulates Calcium Flux to Specify Border Cell Fate during Drosophila oogenesis.

    PubMed

    Sahu, Aresh; Ghosh, Ritabrata; Deshpande, Girish; Prasad, Mohit

    2017-01-01

    Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. Here we report functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Our data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. We show that Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis has uncovered that Inx2 also regulates a calcium flux that transmits across the follicle cells. We propose that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together our data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification.

  12. Functional heterologous gap junctions in Fundulus ovarian follicles maintain meiotic arrest and permit hydration during oocyte maturation.

    PubMed

    Cerdá, J L; Petrino, T R; Wallace, R A

    1993-11-01

    The physiological significance of heterologous gap junctions between granulosa cells and the oocyte was investigated in late vitellogenic ovarian follicles of the teleost Fundulus heteroclitus. Lucifer Yellow injected into the oocyte readily passed to the overlying granulosa cells, demonstrating effective dye-coupling. Passage of the fluorescent dye, and hence intercellular communication, was inhibited both by the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and by 1-octanol, known uncouplers of gap junctions in a variety of invertebrate and vertebrate cell types. Octanol alone also initiated resumption of meiosis in follicle-enclosed oocytes, indicating that granulosa cells normally maintain meiotic arrest, as apparently occurs in mammalian and amphibian follicles. Both PMA and octanol also consistently inhibited the hydration process that normally accompanies meiotic maturation. These results support a previously suggested hypothesis that K+, which is the primary osmotic effector for oocyte hydration, is translocated via gap junction from granulosa cells to the maturing oocyte.

  13. A Gap Junction Protein, Inx2, Modulates Calcium Flux to Specify Border Cell Fate during Drosophila oogenesis

    PubMed Central

    Ghosh, Ritabrata; Deshpande, Girish

    2017-01-01

    Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. Here we report functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Our data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. We show that Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis has uncovered that Inx2 also regulates a calcium flux that transmits across the follicle cells. We propose that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together our data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification. PMID:28114410

  14. Connexins and gap junctions in the inner ear--it's not just about K⁺ recycling.

    PubMed

    Jagger, Daniel J; Forge, Andrew

    2015-06-01

    Normal development, function and repair of the sensory epithelia in the inner ear are all dependent on gap junctional intercellular communication. Mutations in the connexin genes GJB2 and GJB6 (encoding CX26 and CX30) result in syndromic and non-syndromic deafness via various mechanisms. Clinical vestibular defects, however, are harder to connect with connexin dysfunction. Cx26 and Cx30 proteins are widely expressed in the epithelial and connective tissues of the cochlea, where they may form homomeric or heteromeric gap junction channels in a cell-specific and spatiotemporally complex fashion. Despite the study of mutant channels and animal models for both recessive and dominant autosomal deafness, it is still unclear why gap junctions are essential for auditory function, and why Cx26 and Cx30 do not compensate for each other in vivo. Cx26 appears to be essential for normal development of the auditory sensory epithelium, but may be dispensable during normal hearing. Cx30 appears to be essential for normal repair following sensory cell loss. The specific modes of intercellular signalling mediated by inner ear gap junction channels remain undetermined, but they are hypothesised to play essential roles in the maintenance of ionic and metabolic homeostasis in the inner ear. Recent studies have highlighted involvement of gap junctions in the transfer of essential second messengers between the non-sensory cells, and have proposed roles for hemichannels in normal hearing. Here, we summarise the current knowledge about the molecular and functional properties of inner ear gap junctions, and about tissue pathologies associated with connexin mutations.

  15. Vitamin D3 regulates the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

    PubMed

    Kelsey, Linda; Katoch, Parul; Ray, Anuttoma; Mitra, Shalini; Chakraborty, Souvik; Lin, Ming-Fong; Mehta, Parmender P

    2014-01-01

    1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions.

  16. 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.

  17. 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.

  18. Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host

    PubMed Central

    Jäderstad, Johan; Jäderstad, Linda M.; Li, Jianxue; Chintawar, Satyan; Salto, Carmen; Pandolfo, Massimo; Ourednik, Vaclav; Teng, Yang D.; Sidman, Richard L.; Arenas, Ernest; Snyder, Evan Y.; Herlenius, Eric

    2010-01-01

    How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication. PMID:20147621

  19. Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host.

    PubMed

    Jäderstad, Johan; Jäderstad, Linda M; Li, Jianxue; Chintawar, Satyan; Salto, Carmen; Pandolfo, Massimo; Ourednik, Vaclav; Teng, Yang D; Sidman, Richard L; Arenas, Ernest; Snyder, Evan Y; Herlenius, Eric

    2010-03-16

    How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

  20. Changing patterns of gap junctional intercellular communication and connexin distribution in mouse epidermis and hair follicles during embryonic development.

    PubMed

    Choudhry, R; Pitts, J D; Hodgins, M B

    1997-12-01

    In the mouse embryo between embryonic days 12 (E12) and 16, regular arrays of epidermal placodes on the mystacial pad develop into whisker follicles. This system was chosen for analysis of gap junctional intercellular communication during differentiation. The patterns of communication were studied by microinjection of the tracers Lucifer yellow-CH (LY-CH) and neurobiotin (NB), while immunofluorescent staining was used to study distribution of connexins 26 and 43. Extensive communication was seen between keratinocytes in developing hair pegs or, in later-stage hair follicles, in the germinative matrix. Coupling between adjacent hair pegs via interfollicular epidermis was not observed. Coupling also became restricted as follicular cells differentiated to form outer root sheath, inner root sheath, and hair shaft. Extensive gap junctional coupling is characteristic of keratinocytes that are rapidly proliferating (as in hair pegs and germinative matrix). Follicular keratinocytes commence differentiation shortly before restriction of gap junctional coupling becomes evident. Dermal mesenchymal cells undergoing different modes of differentiation also exhibit differences in gap junctional coupling, as evidenced by poor transfer of LY-CH between cells in dermal condensations of hair follicles compared with extensive transfer elsewhere in the dermis. LY-CH and NB were not transferred between epidermal or follicular epithelium and mesenchyme, arguing against a direct role for gap junctions permeable to known second messenger molecules or nucleotides in epithelial-mesenchymal interactions in this system. The distribution of connexins 26 and 43 in epidermis and hair follicles changed during differentiation but there was no correlation with changing patterns of dye transfer, indicating an unexpected degree of complexity in the relationship between gap junctional intercellular communication and connexin protein distribution during development.

  1. 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

  2. Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

    PubMed Central

    Basu, Rahul; Banerjee, Kaveri; Bose, Abhishek

    2015-01-01

    ABSTRACT Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. IMPORTANCE We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition

  3. Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo.

    PubMed

    Basu, Rahul; Banerjee, Kaveri; Bose, Abhishek; Das Sarma, Jayasri

    2015-12-16

    Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction

  4. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells.

    PubMed

    Cheng, B; Zhao, S; Luo, J; Sprague, E; Bonewald, L F; Jiang, J X

    2001-02-01

    Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. "Scrape-loading" dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the

  5. Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor.

    PubMed

    Cherian, Priscilla P; Cheng, Benxu; Gu, Sumin; Sprague, Eugene; Bonewald, Lynda F; Jiang, Jean X

    2003-10-31

    Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components

  6. ATP counteracts the rundown of gap junctional channels of rat ventricular myocytes by promoting protein phosphorylation.

    PubMed

    Verrecchia, F; Duthe, F; Duval, S; Duchatelle, I; Sarrouilhe, D; Herve, J C

    1999-04-15

    1. The degree of cell-to-cell coupling between ventricular myocytes of neonatal rats appeared well preserved when studied in the perforated version of the patch clamp technique or, in double whole-cell conditions, when ATP was present in the patch pipette solution. In contrast, when ATP was omitted, the amplitude of junctional current rapidly declined (rundown). 2. To examine the mechanism(s) of ATP action, an 'internal perfusion technique' was adapted to dual patch clamp conditions, and reintroduction of ATP partially reversed the rundown of junctional channels. 3. Cell-to-cell communication was not preserved by a non-hydrolysable ATP analogue (5'-adenylimidodiphosphate, AMP-PNP), indicating that the effect most probably did not involve direct interaction of ATP with the channel-forming proteins. 4. An ATP analogue supporting protein phosphorylation but not active transport processes (adenosine 5'-O-(3-thiotriphosphate), ATPgammaS) maintained normal intercellular communication, suggesting that the effect was due to kinase activity rather than to altered intracellular Ca2+. 5. A broad spectrum inhibitor of endogenous serine/threonine protein kinases (H7) reversibly reduced the intercellular coupling. A non-specific exogenous protein phosphatase (alkaline phosphatase) mimicked the effects of ATP deprivation. The non-specific inhibition of endogenous protein phosphatases resulted in the preservation of substantial cell-to-cell communication in ATP-free conditions. 6. The activity of gap junctional channels appears to require both the presence of ATP and protein kinase activity to counteract the tonic activity of endogenous phosphatase(s).

  7. Stochastic Model of Gap Junctions Exhibiting Rectification and Multiple Closed States of Slow Gates.

    PubMed

    Snipas, Mindaugas; Kraujalis, Tadas; Paulauskas, Nerijus; Maciunas, Kestutis; Bukauskas, Feliksas F

    2016-03-29

    Gap-junction (GJ) channels formed from connexin (Cx) proteins provide direct pathways for electrical and metabolic cell-cell communication. Earlier, we developed a stochastic 16-state model (S16SM) of voltage gating of the GJ channel containing two pairs of fast and slow gates, each operating between open (o) and closed (c) states. However, experimental data suggest that gates may in fact contain two or more closed states. We developed a model in which the slow gate operates according to a linear reaction scheme, o↔c1↔c2, where c1 and c2 are initial-closed and deep-closed states that both close the channel fully, whereas the fast gate operates between the open state and the closed state and exhibits a residual conductance. Thus, we developed a stochastic 36-state model (S36SM) of GJ channel gating that is sensitive to transjunctional voltage (Vj). To accelerate simulation and el