Receptor activator of NF-kappaB ligand (RANKL) is crucial in osteoclastogenesis but signaling events involved in osteoclast differentiation are far from complete and other signals may play a role in osteoclastogenesis. A more direct pathway for cellular crosstalk is provided by gap junction intercellular channel, which allows adjacent cells to exchange second messengers, ions, and cellular metabolites. Here we have investigated the role of gap junction communication in osteoclastogenesis in mouse bone marrow cultures. Immunoreactive sites for the gap junction protein connexin 43 (Cx43) were detected in the marrow stromal cells and in mature osteoclasts. Carbenoxolone (CBX) functionally blocked gap junction communication as demonstrated by a scrape loading Lucifer Yellow dye transfer technique. CBX caused a dose-dependent inhibition (significant , or = 90 microM) of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells formed in 7- to 8-day marrow cultures ...
The biogenesis of connexins and their assembly into functional gap junction hemichannels (connexons) was studied with the use of a cell-free transcription/translation system. Velocity sedimentation on sucrose gradients showed that a small proportion of connexin (Cx) 26 and Cx32 that were co-translationally translocated into microsomes were oligomers of Cx26 and Cx32. Chemical cross-linking studies showed that these corresponded to hexameric connexons. Reconstitution of connexons synthesized in vitro into liposomes induced permeability properties consistent with the view that open gap junction hemichannels were produced. By using an immunoprecipitation approach, a simultaneous translation of Cx26 and Cx32 incorporated into microsomes resulted in homomeric connexons. However, supplementation of the translation system in vitro with liver Golgi membranes produced heteromeric connexons constructed of Cx32 and Cx26, and also resulted in an increased oligomerization especially of Cx32. All of the ...
During blood vessel assembly, mural cell differentiation is initiated in response to contact-dependent interactions with endothelial cells.5,7 In the present studies, we demonstrate for the first time that functional gap junction channels are established between endothelial cells and the mural cell progenitors that they recruit and are necessary for endothelial-induced mural cell differentiation. We additionally determined that the mechanism by which gap junctions mediate endothelial-induced mural cell differentiation involves the activation of TGF-β, which then induces SRF and mural cell-specific gene expression.. The mesenchymal cells that did not form gap junctions with endothelial cells (Cx43−/−) did not undergo endothelial-induced differentiation toward a mural cell phenotype. Of note, the Cx43−/− cells express low levels of Cx45, although this expression was not evident as channel activity and not adequate to support the formation of functional heterocellular gap junctions with ...
A gap junction may also be called a nexus or macula communicans. When found in neurons or nerves it may also be called an electrical synapse. While an ephapse has some similarities to a gap junction, by modern definition the two are different. Gap junctions are a specialized intercellular connection between a multitude of animal cell-types. They directly connect the cytoplasm of two cells, which allows various molecules, ions and electrical impulses to directly pass through a regulated gate between cells. One gap junction channel is composed of two connexons (or hemichannels), which connect across the intercellular space. Gap junctions are analogous to the plasmodesmata that join plant cells. Gap junctions occur in virtually all tissues of the body, with the exception of adult fully developed skeletal muscle and mobile cell types such as sperm or erythrocytes. Gap junctions, however, are not found in simpler organisms such as sponges and slime molds. In vertebrates, gap junction hemichannels are ...
Reversible down-regulation of gap junctional intercellular communication (GJIC) is proposed to be an important cellular mechanism in tumor promotion. Gap junction function is modified by a variety of tumor promoters, including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of cells with TPA results in the activation and subsequent depletion of the TPA-responsive protein kinase C (PKC) isoforms. TPA-induced degradation of the PKC isoforms α, δ and ϵ was recently shown to occur via the ubiquitin-proteasome pathway. In the present study we investigated the role of the proteasome in the TPA-induced modification of GJIC in IAR20 rat liver epithelial cells. TPA exposure of IAR20 cells induced hyperphosphorylation of gap junction protein connexin43 and inhibition of GJIC. Prolonged TPA treatment induced down-regulation of PKCα, δ and ϵ and a reduction in the total PKC activity, which was associated with recovery of GJIC. Co-treatment of IAR20 cells with TPA and the ...
TY - JOUR. T1 - Gap junctional channels regulate acid secretion in the mammalian gastric gland. AU - Radebold, K.. AU - Horakova, E.. AU - Gloeckner, J.. AU - Ortega, G.. AU - Spray, David C.. AU - Vieweger, H.. AU - Siebert, K.. AU - Manuelidis, L.. AU - Geibel, J. P.. PY - 2001/10/1. Y1 - 2001/10/1. N2 - Gap junction channels are regarded as a primary pathway for intercellular message transfer, including calcium wave propagation. Our study identified two gap junctional proteins, connexin26 and connexin32, in rat gastric glands by RT-PCR, Western blot analysis, and immunofluorescence. We demonstrated a potential physiological role of the gap junctional channels in the acid secretory process using the calcium indicator fluo-3, and microinjection of Lucifer Yellow. Application of gastrin (10-7 M) to the basolateral membrane resulted in the induction of uniphasic calcium signals in adjacent parietal cells. In addition, single parietal cell microinjections in intact glands with the cell-impermeant ...
The mechanism by which the change in gap-junctional distribution influences the location and characteristics of the reentrant circuit has yet to be determined. The abnormal redistribution of gap junctions to the lateral interfaces between myocytes might be expected to enhance side-to-side coupling, thereby improving transverse conduction and reducing (rather than increasing) the arrhythmogenicity of the tissue by reducing the degree of anisotropy. The redistribution should, however, be considered in the context of other changes of electrophysiological significance in the infarct border zone myocytes.14 15 43 Although the functional status of the gap junctions in these border zone cells is unknown, the results of in vitro studies would suggest that the hypoxia, acidosis, and hypercalcemia that may exist in this tissue would attenuate gap-junctional coupling44 and reduce conductance particularly in the transverse direction.45 46 Indeed, one might speculate that the redistribution of gap junctions ...
TY - JOUR. T1 - Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary. AU - Okuma, A.. AU - Kuraoka, A.. AU - Iida, H.. AU - Inai, T.. AU - Wasano, K.. AU - Shibata, Y.. PY - 1996/7. Y1 - 1996/7. N2 - The expression and localization of gap junction family proteins (connexins) were examined in nonstimulated and gonadotrophin-stimulated ovarian follicles of immature rats. Immunoblot and RNA blot analysis showed the presence of connexin (Cx) 43, Cx40 and Cx45 in ovarian tissue. Of these connexin proteins, Cx43 and Cx45 were identified by immunofluorescent microscopy between granulosa cells in characteristic expression patterns related to follicular developmental stages, while Cx40 was not expressed in granulosa cells but was detected in blood vessels in ovarian stroma. In some plaques of gap junction between granulosa cells, Cx45 was found to be colocalized with Cx43. In immunofluorescent microscopy, the expression of Cx43 was ...
Early descriptions of "gap junctions" and "connexons" did not refer to them as such and many other terms were used. It is likely that "synaptic disks"[116] were an accurate reference to gap junction plaques. While the detailed structure and function of the connexon was described in a limited way at the time the gross "disk" structure was relatively large and easily seen by various TEM techniques. Disks allowed researchers using TEM to easily locate the connexons contained within the disk like patches in vivo and in vitro. The disk or "plaque" appeared to have structural properties different from those imparted by the connexons alone.[25] It was thought that if the area of membrane in the plaque transmitted signals the area of membrane would have to be sealed in some way to prevent leakage.[117] Later studies showed gap junction plaques are home to non-connexin proteins making the modern usage of the terms "gap junction" and "gap junction plaque" non-interchangeable as the area of the gap ...
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 ...
My research interests focus on gap junctions which are involved in cell-to-cell communication. Regulating the chemical and physical properties of gap junctions are the different connexin proteins. Unique communication compartments can be formed when gap junctions are assembled from various connexins.. Presently, I am examining the implications of gap junctions on cell differentiation and development using the testis as a model. Organized in the seminiferous tubule and supported by Sertoli cells are some 63 different germ cell types. The germ cells are arranged and organized in the seminiferous epithelium for an ordered development and differentiation into spermatozoa. We are currently determining gap junctions role in the formation of specific communication compartments and how gap junctions regulate and support specific spermatogenic cells. Gap junction assembly, connexin composition, and the chemical and physical properties of homotypic - heterotypic gap junctions will be examined ...
Introduction: Metastasis involves the emigration of tumor cells through the vascular endothelium, a process also known as diapedesis. The molecular mechanisms regulating tumor cell diapedesis are poorly understood, but may involve heterocellular gap junctional intercellular communication (GJIC) between tumor cells and endothelial cells. Method: To test this hypothesis we expressed connexin 43 (Cx43) in GJIC-deficient mammary epithelial tumor cells (HBL100) and examined their ability to form gap junctions, establish heterocellular GJIC and migrate through monolayers of human microvascular endothelial cells (HMVEC) grown on matrigel-coated coverslips. Results: HBL100 cells expressing Cx43 formed functional heterocellular gap junctions with HMVEC monolayers within 30 minutes. In addition, immunocytochemistry revealed Cx43 localized to contact sites between Cx43 expressing tumor cells and endothelial cells. Quantitative analysis of diapedesis revealed a two-fold increase in diapedesis of Cx43 expressing
PURPOSE To establish an electrical fingerprint for the gap junction channels between mammalian lens epithelial cells. METHODS The double whole cell patch clamp technique was applied to isolated cell pairs obtained from mouse lens epithelium and a continuous cell line of lens epithelial cells derived from the sheep lens (SLE 2.1). RESULTS The junctional conductance in mouse lens epithelial cells and in cultured SLE 2.1 cells was found to be moderately voltage dependent. SLE 2.1 cells were analyzed in more detail. The voltage dependence could be described by a Boltzmann distribution with Vo = +/- 63.1 mV and Gmin = 0.34. In cell pairs that exhibited spontaneously low junctional conductance, single channel events could be distinguished. Single gap junction channel currents had a linear current-voltage relationship. A frequency histogram of single channel conductances from eight cell pairs had three major peaks of 35, 60 and 97 pS. CONCLUSION The electrical properties of gap junction channels
TY - JOUR. T1 - Glycosaminoglycans and proteoglycans induce gap junction expression and restore transcription of tissue‐specific mRNAs in primary liver cultures. AU - Fujita, Michiyasu. AU - Spray, David C.. AU - Choi, Haing. AU - Saez, Juan C.. AU - Watanabe, Tohru. AU - Rosenberg, Larry C.. AU - Hertzberg, Elliott L.. AU - Reid, Lola M.. PY - 1987/1/1. Y1 - 1987/1/1. N2 - Normal rat hepatocytes maintained on tissue culture plastic and in serum‐supplemented medium lose their gap junctions within 12 hr and expression of their tissue‐specific functions within 24 to 72 hr. The gap junctions are lost via internalization and degradation, and the differentiated functions due to loss of synthesis and to rapid degradation of tissue‐specific mRNAs. Near normal levels of tissue‐specific mRNAs can be achieved by stabilization of the mRNAs but not by transcription (for most genes), if the cells are cultured in a serum‐free, hormonally defined medium and on substrata of tissue culture plastic, ...
Gap junctions are clusters of specialized intercellular channels that regulate the direct exchange of ions and various hydrophilic cellular metabolites that are smaller than 1000 Da, a process known as gap junctional intercellular communication (GJIC) (Alexander and Goldberg, 2003). Two inter-docked connexons (hemichannels), one from each of two apposing cells, form a functional gap junction channel. Each connexon is composed of six oligomerized connexin (Cx) subunits, and, to date, the connexin family consists of 21 members in human (Söhl and Willecke, 2003; Söhl and Willecke, 2004). Interestingly, the primary function of gap junction channels is to facilitate intercellular communication; however, hemichannels have also been reported to exist and function at the cell surface in an undocked state, permitting the transfer of molecules between extracellular and intracellular environments (Anselmi et al., 2008; Burra and Jiang, 2011; Tong et al., 2007). Hemichannels that are formed from single or ...
Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells.
Gap junctions contain intercellular channels that allow direct communication between the cytosolic compartments of adjacent cells. Each gap junction channel is formed by docking of two hemichannels, each containing six connexins, contributed by each neighboring cell. These channels permit the direct transfer of small molecules including ions, amino acids, nucleotides, second messengers and other metabolites between adjacent cells. Gap junctional communication is essential for many physiological events, including embryonic development, electrical coupling, metabolic transport, apoptosis, and tissue homeostasis. Communication through Gap Junction is sensitive to a variety of stimuli, including changes in the level of intracellular Ca2+, pH, transjunctional applied voltage and phosphorylation/dephosphorylation processes. This figure represents the possible activation routes of different protein kinases involved in Cx43 and Cx36 phosphorylation ...
Gap junctions contain intercellular channels that allow direct communication between the cytosolic compartments of adjacent cells. Each gap junction channel is formed by docking of two hemichannels, each containing six connexins, contributed by each neighboring cell. These channels permit the direct transfer of small molecules including ions, amino acids, nucleotides, second messengers and other metabolites between adjacent cells. Gap junctional communication is essential for many physiological events, including embryonic development, electrical coupling, metabolic transport, apoptosis, and tissue homeostasis. Communication through Gap Junction is sensitive to a variety of stimuli, including changes in the level of intracellular Ca2+, pH, transjunctional applied voltage and phosphorylation/dephosphorylation processes. This figure represents the possible activation routes of different protein kinases involved in Cx43 and Cx36 phosphorylation ...
Objective: Gap junctions (formed by connexins, Cx) are important for functional coordination of cells in the vascular wall. However, little is known about their physiological regulation in this tissue. We examined the effects of nitric oxide (NO), an important mediator of vasomotion, wound healing and angiogenesis, on the formation of gap junctions in endothelial cells (human umbilical vein endothelial cells, HUVEC). Methods: Flow cytometry was used to determine dye transfer through newly formed gap junctions between acutely coincubated HUVECs. Parallel experiments in wild-type HeLa cells (no connexins) and transfected HeLa cells exclusively expressing Cx43, Cx40 or Cx37 were performed to determine the specific role of Cx subtypes. The intracellular distribution of Cx40 was examined after fractionation with triton by Western blotting. Intracellular levels of cGMP and cAMP were measured by radioimmunoassay. Results: The NO donor SNAP (1 μM) enhanced gap-junctional coupling in HUVECs by about ...
Connexin 47 (Cx47), and to a lesser extent Cx32, are assembled into Gap Junctions (GJs) and couple oligodendrocytes mainly to astrocytes via heterotypic GJs and to other oligodendrocytes via homotypic GJs. In the demyelinating and inflammatory disease Multiple Sclerosis (MS) these oligodendrocyte connexins appear disrupted. Recently published studies in our lab utilizing mice with Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS, demonstrates that the absence of Cx32 or Cx47 affects the severity and progression of the disease and alters the profile of several CNS-inflammation-related cytokines. From these 64 cytokines tested in wild type (WT) EAE, Cx32-knockout (KO) EAE, and Cx47-KO EAE mice, we selected the most relevant and altered molecules, GM-CSF, G-CSF, CCL2, and VCAM-1 for further testing in the same mouse genotypes. Their expression was analyzed at 7, 12, and 24 days post injection (dpi) using fluorescent microscopy, real-time PCR, and Western blot analysis. This ...
TY - JOUR. T1 - Intercellular communication and human hepatocellular carcinoma. AU - Carruba, Giuseppe. AU - Cocciadiferro, Letizia. AU - Bellavia, Vincenzo. AU - Rizzo, Sergio. AU - Tsatsanis, Christos. AU - Spandidos, Demetrios. AU - Muti, Paola. AU - Smith, Colin. AU - Mehta, Parmender P. AU - Castagnetta, Luigi. PY - 2004/1/1. Y1 - 2004/1/1. N2 - We have previously reported that gap junction-mediated intercellular communication (GJIC) can be restored in junctionally deficient human prostate epithelial cells, also suggesting that GJIC activity is regulated by estrogen. In the present work, we report studies on sex steroid regulation of GJIC and proliferative activity in both nontumoral (Chang liver, CL) and malignant (HepG2, Huh7) human liver cells. Junctional activity and liver cell growth were measured using the scrape-loading/dye-transfer (SL/DT) and the MTS assay, respectively. Using the SL/DT, only Huh7 cells exhibited a moderate degree of Junctional activity in basic conditions, while ...
There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.
Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis ...
Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXXØ signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXØ motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the ...
Gap junction protein that acts as a regulator of bladder capacity. A gap junction consists of a cluster of closely packed pairs of transmembrane channels, the connexons, through which materials of low MW diffuse from one cell to a neighboring cell. May play a critical role in the physiology of hearing by participating in the recycling of potassium to the cochlear endolymph. Negative regulator of bladder functional capacity: acts by enhancing intercellular electrical and chemical transmission, thus sensitizing bladder muscles to cholinergic neural stimuli and causing them to contract. May play a role in cell growth inhibition through the regulation of NOV expression and localization. Plays an essential role in gap junction communication in the ventricles (By similarity).
Gap junction protein that acts as a regulator of bladder capacity. A gap junction consists of a cluster of closely packed pairs of transmembrane channels, the connexons, through which materials of low MW diffuse from one cell to a neighboring cell. May play a critical role in the physiology of hearing by participating in the recycling of potassium to the cochlear endolymph. Negative regulator of bladder functional capacity: acts by enhancing intercellular electrical and chemical transmission, thus sensitizing bladder muscles to cholinergic neural stimuli and causing them to contract. May play a role in cell growth inhibition through the regulation of NOV expression and localization. Plays an essential role in gap junction communication in the ventricles (By similarity).
Evidence implicating alterations in connexin43 gap junctions in arrhythmogenesis in different models of human heart disease has steadily accumulated.20 21 22 26 29 30 The novel findings of the present study are that (1) a reduction in connexin43 gap junctions occurs in hibernating myocardium beyond that seen in reversible ischemia, and (2) a specific feature characterizing hibernating myocardium is a loss of the population of large gap junctions at the disk periphery. These changes seen by laser scanning immunoconfocal microscopy were subsequently confirmed by quantification with PC image analysis. The use of quantitative immunofluorescence for measurement of gap junction size has been previously validated with a polyclonal anti-connexin43 antibody in rat left ventricular tissue by comparison of measurements from immunoconfocal (0.53 μm) and freeze-fracture (0.57 μm) electron microscopy.28 In the present study, quantification of images obtained with a different primary antibody yielded a mean ...
Our results demonstrate changes of gap junction channel characteristics and alterations in the pathways of intercellular communication in the organ of Corti during postnatal development. The characteristics of early postnatal GJIC bear little resemblance to those in the hearing cochlea. These observations have implications for the interpretation of studies of GJIC in normal hearing and disease. We have provided functional evidence for the existence of gap junction channels comprising heteromeric Cx26/Cx30 connexons in native cochlear tissue in hearing animals based on the selective transfer of diagnostic dyes, in conjunction with the colocalization of Cx26 and Cx30 within supporting cells. We found evidence for Cx26-only channels (i.e., LY permeable) only in peripheral supporting cells within the organ of Corti in hearing animals (supplemental Fig. 4, available at www.jneurosci.org as supplemental material). Furthermore, we have provided functional and anatomical evidence that is consistent with ...
Guys, Ive uploaded my disease stuff except im having problems with uploading the references and pictures. WIll try again later. Ive also added a few things to our glossary. Anyway hope youve all had a good break! --Elizabeth Blanchard 10:28, 30 April 2011 (EST) I found another article concerning disease that might be of use: Gap-Junction Channels Dysfunction in Deafness and Hearing Loss I wouldnt put the abstract since its too long. [1] --z3283837 23:00, 26 April 2011 (EST) Hey guys, I am kind of finished with my part for the intro and the function although it needs a little bit of touch up. Anyway, while researching, I just found a few review articles that might be helpful. Whoever is doing the location of gap junctions, this article below gives you an overview of the expression patterns of different connexins in different tissues. Diversity and properties of connexin gap junction channels Gap junction channels are composed of two apposing hemichannels (connexons) in the contiguous cells ...
Guys, Ive uploaded my disease stuff except im having problems with uploading the references and pictures. WIll try again later. Ive also added a few things to our glossary. Anyway hope youve all had a good break! --Elizabeth Blanchard 10:28, 30 April 2011 (EST) I found another article concerning disease that might be of use: Gap-Junction Channels Dysfunction in Deafness and Hearing Loss I wouldnt put the abstract since its too long. [1] --z3283837 23:00, 26 April 2011 (EST) Hey guys, I am kind of finished with my part for the intro and the function although it needs a little bit of touch up. Anyway, while researching, I just found a few review articles that might be helpful. Whoever is doing the location of gap junctions, this article below gives you an overview of the expression patterns of different connexins in different tissues. Diversity and properties of connexin gap junction channels Gap junction channels are composed of two apposing hemichannels (connexons) in the contiguous cells ...
Purpose: Cancer patients are often concurrently treated with analgesics and antineoplastic drugs, yet the influence of analgesic agents on therapeutic activity of antineoplastic drugs is largely unexplored. This study investigates the effects of three commonly used analgesics, which produce analgesia by different mechanisms, on cytotoxicity induced by cisplatin, a widely used antitumor agent, and the relation between those effects and modulation of gap junction function by the analgesics.. Experimental Design: The role of gap junctions in the modulation of cisplatin toxicity is explored by manipulation of connexin expression, and gap junction presence and function, using clinically relevant concentrations of the analgesics and cisplatin.. Results: Short-term exposure of transformed cells to cisplatin reduced the clonogenic survival in low-density cultures (without gap junction formation) and in high density (with gap junction formation), but the toxic effect was greater at high density. In the ...
Ng, FS, Lyon, AR, Shadi, IT, Chang, ETY, Chowdhury, RA, Dupont, E and Peters, NS (2010) GAP JUNCTIONAL UNCOUPLING WITH CARBENOXOLONE SLOWS CONDUCTION AND INCREASES VULNERABILITY TO VENTRICULAR ARRHYTHMIAS IN STRUCTURALLY NORMAL HEARTS: AN OPTICAL MAPPING STUDY In: Annual Conference and Exhibition of the British-Cardiovascular-Society, 2010-06-07 - 2010-06-09, Manchester, ENGLAND. Ng, FS, Lyon, AR, Shadi, IT, Chang, ETY, Chowdhury, RA, Dupont, E and Peters, NS (2010) MODULATION OF GAP JUNCTIONAL COUPLING AS AN ANTI-ARRHYTHMIC STRATEGY TO PREVENT REPERFUSION VENTRICULAR ARRHYTHMIAS In: Annual Conference and Exhibition of the British-Cardiovascular-Society, 2010-06-07 - 2010-06-09, Manchester, ENGLAND. Dhillon, PS, Gray, R, Kojodjojo, P, Jabr, R, Chowdhury, RA, Fry, CH and Peters, NS (2009) The Relationship Between Gap Junction Conductance and Conduction Velocity in Intact Myocardium In: 82nd Scientific Session of the American-Heart-Association, 2009-11-14 - 2009-11-18, Orlando, FL. Dhillon, P, ...
The molecular basis of gap junctions in invertebrates has been a mystery for decades. Connexin proteins were first identified in the 1970s as the structural components of gap junctions in vertebrates (Goodenough, 1974). For more than 20 years, the term `connexin became synonymous with gap junctions, whereas efforts to find the homologous connexin in invertebrates were unsuccessful (Phelan et al., 1998). It was only in 1998 that the gene family encoding invertebrate gap-junction proteins, innexins, was first confirmed in Drosophila (Phelan et al., 1998). To date, eight innexin genes have been found in the fruit fly, and 25 innexin genes in C. elegans (Phelan, 2005).. In the present study we identify six innexin-like genes in Aedes (Table 5). The neighbor-joining tree (Fig. 9) shows that most of the innexins in Aedes are still closely related to their Drosophila homologues despite the 250 million years of evolution that separate the mosquito from the fruit fly (Severson et al., 2004). The strong ...
Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease In gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Confocal microscopy data indicates that the two proteins are within physical proximity allowing for a potential kinase-substrate interaction. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43 we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially-purified Cx43 was phosphorylated on ...
Heart failure (HF), whether nonischemic or ischemic, is associated with a nearly 50% incidence of sudden death, primarily from ventricular tachycardia (VT) degenerating to ventricular fibrillation (VF).1 Whereas VT in nonischemic HF initiates primarily by a nonreentrant mechanism,2 myocardium from patients with idiopathic dilated cardiomyopathy (IDCM) exhibits nonuniform anisotropy, slow conduction, and conduction block3 that could underlie reentry during the transition from VT to VF. Conduction slowing could arise from decreased depolarizing currents and/or decreased gap junctional coupling.4 However, the degree of slow conduction and block in failing myocardium appears to be out of proportion to the changes in active membrane properties.5 Moreover, LV myocytes from an animal model of nonischemic HF exhibit markedly decreased gap junctional conductance.6 Thus, alterations in intercellular coupling involving cardiac gap junctions may underlie slow conduction in nonischemic HF.. Gap junctions are ...
New immunolocalization data put the role of the lens MP26 (MIP) protein in a new perspective. During maturation of lens fibre cells, MIP is found to associate specifically with two structures, gap junctions and cell interlocking processes (known as ball and socket domains). It is significant that the zone in which these associations are most striking is discrete, coinciding with the zone of rapidly enlarging junctional plaques and newly forming ball and socket domains. Observation of domain-specific interactions of MIP with forming gap junctions and ball and socket domains suggests that MIP may be involved in the formation of close membrane appositions. Furthermore, previous ambiguities in the literature over the presence of MIP in gap junctions are clarified by the knowledge that, in situ, MIP associates strongly with gap junctions for only a brief period (with less than about 5% of all lens gap junctions at any one time) during the assembly of junctional plaques. ...
Model of the role of FGF in establishing regional differences in gap junction-mediated intercellular communication in the lens. (A) The concentration of FGF i
Intracellular calcium (Ca2+) concentration exhibits periodic oscillations in vascular smooth muscle cells. This is thought to result from Ca2+ release from intracellular stores, due to inositol triphosphate and ryanodine-sensitive channel activation. This activation has been shown to result in either Ca2+ "sparks", highly localized calcium increases, or "waves", global Ca2+ increase that propagates the length of the cell.[3] To allow vasomotion to occur, synchronization must occur between the individual oscillations, resulting in global calcium synchronization and vessel tone oscillation.[4] Gap junctions are thought to play a large role in this synchronization, as application of gap junction blockers has been shown to abolish vasomotion, indicating a critical role.[5] Due to regional variations in gap junction distribution and coupling (homocellular vs. heterocellular) several hypotheses have been suggested to account for vasomotion occurrence. The "classic" mechanism of vasomotion generation ...
In the present study, we provide experimental evidence that gap junctions/hemichannels are involved in the apical phase of interkinetic nuclear migration in neural precursors. Our data suggest that regulation of apically directed interkinetic nuclear migration by intracellular Ca2+ signaling via both ATP release and Ca2+-mobilizing messenger diffusion may be an important mechanism by which functional gap junctions/hemichannels maintain the neural progenitor pool during their division.. Cytosolic Ca2+ signaling has previously been implicated in several aspects of nervous system development, including cell proliferation (Weissman et al., 2004; Pearson et al., 2005), differentiation (Gu and Spitzer, 1995), migration (Komuro and Rakic, 1993), neurite outgrowth, and growth cone behavior (Gomez and Spitzer, 1999). In the present study, we confirmed the existence of Ca2+ signaling fluctuations in neural precursors of the VZ/SVZ (Owens and Kriegstein, 1998; Weissman et al., 2004). However, in addition, ...
When DArcy Wentworth Thompsons On Growth and Form was published 100 years ago, it raised the question of how biological forms arise during development and across evolution. In light of the advances in molecular and cellular biology since then, a succinct modern view of the question states: how do genes encode geometry? Our new special issue is packed with articles that use mathematical and physical approaches to gain insights into cell and tissue patterning, morphogenesis and dynamics, and that provide a physical framework to capture these processes operating across scales.. Read the Editorial by guest editors Thomas Lecuit and L. Mahadevan, as they provide a perspective on the influence of DArcy Thompsons work and an overview of the articles in this issue.. ...
Gap junctions provide direct electrical and biochemical communication between cardiomyocytes in the heart. Connexin40 (Cx40) is the major connexin in the atria of the heart and little is known regarding its regulation. Thus, the goal was to investigate the regulation of Cx40 in both physiological and pathophysiological conditions. The first objective of this thesis was to determine whether Cx40 gap junctions were regulated by â-adrenergic receptor activation. Cx40 has previously been shown to be acutely activated by cAMP, this cAMP-induced increase in Cx40-mediated cell-to-cell dye transfer has been shown to be effected through the â-adrenergic receptor-adenylyl cyclase- Protein Kinase A (PKA) pathway in Cx40-transfected HeLa cells. The second objective of this thesis was to determine whether Cx40 gap junctions were regulated by intracellular Ca2+ concentration ([Ca2+]i ). [Ca2+]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca2+]o from 1.8 mM to 21.8 mM.
Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is ...
While a number of different gap junction proteins have now been identified, hepatic gap junctions are unique in being the first demonstrated case where two homologous, but distinct, proteins (28,000 and 21,000 Mr) are found within a single gap junctional plaque (Nicholson, B. J., R. Dermietzel, D. Teplow, O. Traub, K. Willecke, and J.-P. Revel. 1987. Nature [Lond.]. 329:732-734). The cDNA for the major 28,000-Mr component has been cloned (Paul, D. L. 1986. J. Cell Biol. 103:123-134) (Kumar, N. M., and N. B. Gilula. 1986. J. Cell Biol. 103:767-776) and, based on its deduced formula weight of 32,007, has been designated connexin 32 (or Cx32 as used here). We now report the selection and characterization of clones for the second 21,000-Mr protein using an oligonucleotide derived from the amino-terminal protein sequence. Together the cDNAs represent 2.4 kb of the single 2.5-kb message detected in Northern blots. An open reading frame of 678 bp coding for a protein with a calculated molecular mass of ...
Effects of second messengers on gap junctional intercellular communication of ovine luteal cells throughout the estrous cycle.: Corpora lutea (CL) from Days 5,
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, ...
Principal Investigator:TOMOYOSE Taiki, Project Period (FY):2003 - 2004, Research Category:Grant-in-Aid for Scientific Research (C), Section:一般, Research Field:Surgical dentistry
Purpose: : The purpose of this study is to determine the PKCγ phosphorylation site on Cx50 in lens epithelial cells and subsequent functional results of phosphorylation. Methods: : Mutation (S430A) was introduced into the wild type Cx50:EGFP by site-directed mutagenesis. Wild-type and mutated (S430A) Cx50 were transfected into 80% confluent N/N lens epithelial cells, and stably transfected cells were selected in DMEM media. PKCγ was activated by phorbol-12 myristate 13 acetate (TPA, 200nM). Expression and localization of wild type and mutated Cx50-EGFP fusion proteins before and after TPA treatment were measured by confocal microscopy. Cell surface Cx50 gap junction plaques were immuno-labeled and counted by confocal microscopy. Co-localization of Cx50 and PKCγ was determined by co-immunoprecipitation. Phosphorylation of Cx50-S430 was determined by western blotting with anti-phosphoserine antibodies and functional effects were measured by gap junction plaque assembly-disassembly. Results: : ...
Cardiac myocytes and fibroblasts form extensive networks in the heart, with numerous anatomical contacts between cells. Fibroblasts, obligatory components of the extracellular matrix, represent the majority of cells in the normal heart, and their number increases with aging and during disease. The myocyte network, coupled by gap junctions, is generally believed to be electrically isolated from fibroblasts in vivo. In culture, however, the heterogeneous cell types form functional gap junctions, which can provide a substrate for electrical coupling of distant myocytes, interconnected by fibroblasts only. Whether similar behavior occurs in vivo has been the subject of considerable debate. Recent electrophysiological, immunohistochemical, and dye-coupling data confirmed the presence of direct electrical coupling between the two cell types in normal cardiac tissue (sinoatrial node), and it has been suggested that similar interactions may occur in post-infarct scar tissue. Such heterogeneous cell ...
Fibroblasts play a significant role in the development of electrical and mechanical dysfunction of the heart; however, the underlying mechanisms are only partially understood. One widely studied mechanism suggests that fibroblasts produce excess extracellular matrix, resulting in collagenous septa t …
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