scholarly journals Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763 ◽  
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells

1997 ◽  
Vol 110 (4) ◽  
pp. 497-504 ◽  
Author(s):  
D. Cao ◽  
G. Lin ◽  
E.M. Westphale ◽  
E.C. Beyer ◽  
T.H. Steinberg
Keyword(s):  
Gap Junction ◽  
Islet Cells ◽  
Cytosolic Calcium ◽  
Calcium Waves ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Junction Protein ◽  
Insulin Secreting Cells ◽  
Gap Junction Protein ◽  
Gap Junctional

Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.

Shapes of astrocyte networks in the juvenile brain

2006 ◽  
Vol 2 (1) ◽  
pp. 3-14 ◽  
Author(s):  
VANESSA HOUADES ◽  
NATHALIE ROUACH ◽  
PASCAL EZAN ◽  
FRANK KIRCHHOFF ◽  
ANNETTE KOULAKOFF ◽  
...  
Keyword(s):  
Gap Junction ◽  
Brain Function ◽  
Connexin 43 ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
High Level ◽  
Gap Junctional ◽  
Neuronal Compartments

The high level of intercellular communication mediated by gap junctions between astrocytes indicates that, besides individual astrocytic domains, a second level of organization might exist for these glial cells as they form communicating networks. Therefore, the contribution of astrocytes to brain function should also be considered to result from coordinated groups of cells. To evaluate the shape and extent of these networks we have studied the expression of connexin 43, a major gap junction protein in astrocytes, and the intercellular diffusion of gap junction tracers in two structures of the developing brain, the hippocampus and the cerebral cortex. We report that the shape of astrocytic networks depends on their location within neuronal compartments in a defined brain structure. Interestingly, not all astrocytes are coupled, which indicates that connections within these networks are restricted. As gap junctional communication in astrocytes is reported to contribute to several glial functions, differences in the shape of astrocytic networks might have consequences on neuronal activity and survival.

scholarly journals Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src.

1988 ◽  
Vol 8 (4) ◽  
pp. 1414-1420 ◽  
Author(s):  
A S Menko ◽  
D Boettiger
Keyword(s):  
Gap Junctions ◽  
Rous Sarcoma Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Junction Protein ◽  
Junction Formation ◽  
Cell Layers ◽  
Sarcoma Virus ◽  
Gap Junction Protein

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

The relationship of gap junctions and compaction in the preimplantation mouse embryo

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 113-118
Author(s):  
David L. Becker ◽  
Catherine Leclerc-David ◽  
Anne Warner
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gap Junctional Communication ◽  
Stage Of Development ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional

In the mouse embryo, gap junctions first appear at the 8-cell stage as compaction is about to take place. Compaction of the embryo is important for the differentiation of the first two cell types; the inner cell mass and the trophectoderm. Our studies examine the contribution of gap junctional communication at this stage of development We have characterised the normal sequence of appearance of gap junction protein and its distribution. The extent of communication as shown by the passage of dye between cells has been recorded in both normal embryos and embryos treated with drugs that influence gap junctional communication. Comparisons have been made with embryos that express a lethal gap junction defect and attempts were made to rescue such embryos by increasing their gap junction communication.

Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src

1988 ◽  
Vol 8 (4) ◽  
pp. 1414-1420
Author(s):  
A S Menko ◽  
D Boettiger
Keyword(s):  
Gap Junctions ◽  
Rous Sarcoma Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Junction Protein ◽  
Junction Formation ◽  
Cell Layers ◽  
Sarcoma Virus ◽  
Gap Junction Protein

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

scholarly journals Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

1990 ◽  
Vol 111 (5) ◽  
pp. 2077-2088 ◽  
Author(s):  
L S Musil ◽  
B A Cunningham ◽  
G M Edelman ◽  
D A Goodenough
Keyword(s):  
Cell Adhesion ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
Gap Junctional Communication ◽  
Competent Cells ◽  
Deficient Cell ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

scholarly journals Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

2005 ◽  
Vol 16 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Joseph P. Stains ◽  
Roberto Civitelli
Keyword(s):  
Protein Kinase ◽  
Gap Junctions ◽  
Gap Junction ◽  
Gene Transcription ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Extracellular Signal ◽  
Gap Junctional

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Iα1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

scholarly journals Blockade of Brain Stem Gap Junctions Increases Phrenic Burst Frequency and Reduces Phrenic Burst Synchronization in Adult Rat

2003 ◽  
Vol 89 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Irene C. Solomon ◽  
Ki H. Chon ◽  
Melissa N. Rodriguez
Keyword(s):  
Gap Junctions ◽  
Brain Stem ◽  
Gap Junction ◽  
Phrenic Nerve ◽  
Respiratory Rhythm ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Nerve Discharge

Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1–5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 μM), 18α-glycyrrhetinic acid (25–100 μM), 18β-glycyrrhetinic acid (25–100 μM), octanol (200–300 μM), or heptanol (200 μM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (∼95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge ( P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T I ( P < 0.01) and T E ( P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a “bell-shaped” or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (≥130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to identify the specific brain stem regions and cell types (i.e., neurons, glia) mediating the observed modulations in phrenic motor output, these findings suggest that gap junction communication modulates generation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents in vitro.

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