scholarly journals Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

2001 ◽  
Vol 12 (4) ◽  
pp. 831-845 ◽  
Author(s):  
Brenda R. Kwak ◽  
Michael S. Pepper ◽  
Daniel B. Gros ◽  
Paolo Meda
Keyword(s):  
Endothelial Cells ◽  
Wound Repair ◽  
Single Cells ◽  
Dominant Negative ◽  
Cell Coupling ◽  
Mechanical Wounding ◽  
Functional Changes ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Gap-junctional communication in a communication-defective and in a communication-competent teratocarcinoma cell line

1985 ◽  
Vol 76 (1) ◽  
pp. 85-95
Author(s):  
C.W. Lo ◽  
D. Fang ◽  
M.L. Hooper
Keyword(s):  
Cell Line ◽  
Coupling Coefficient ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Teratocarcinoma Cell ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Ionic Coupling ◽  
Cell Cell

We examined the gap-junctional communication properties of a communication-defective cell line R5/3 and its communication-competent revertant H2T12. For these studies, we carried out microelectrode impalements to monitor ionic coupling and dye coupling. Our dye-injection experiments revealed that the H2T12 cells are much more efficient in dye coupling than the R5/3 cells. This latter observation is in agreement with the previous finding that the H2T12 cells are much better metabolically coupled than the R5/3 cells. With ionic coupling measurements, however, both cell lines exhibited similar levels of cell-cell coupling. The R5/3 cells demonstrated an ionic coupling coefficient of 0.19 +/− 0.011 (S.E.M.) and H2T12 a coupling coefficient of 0.25 +/− 0.009 (S.E.M.). These results in conjunction with observations from other studies indicate that the different experimental approaches for monitoring gap-junctional communication may have different levels of sensitivity for detecting as opposed to measuring the level of cell-cell coupling.

scholarly journals Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368

2004 ◽  
Vol 167 (3) ◽  
pp. 555-562 ◽  
Author(s):  
Theresa S. Richards ◽  
Clarence A. Dunn ◽  
William G. Carter ◽  
Marcia L. Usui ◽  
John E. Olerud ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Wound Repair ◽  
Time Course ◽  
Phosphorylation Site ◽  
Primary Human Keratinocytes ◽  
Gap Junctional Communication ◽  
Kinase C ◽  
Junctional Communication ◽  
Gap Junctional

Phosphorylation of connexin43 (Cx43) on serine368 (S368) has been shown to decrease gap junctional communication via a reduction in unitary channel conductance. Examination of phosphoserine368 (pS368) in normal human skin tissue using a phosphorylation site–specific antibody showed relatively even distribution throughout the epidermal layers. However, 24 h after wounding, but not at 6 or 72 h, pS368 levels were dramatically increased in basal keratinocytes and essentially lost from suprabasal layers adjacent to the wound (i.e., within 200 μm of it). Scratch wounding of primary human keratinocytes caused a protein kinase C (PKC)-dependent increase in pS368 in cells adjacent to the scratch, with a time course similar to that found in the wounds. Keratinocytes at the edge of the scratch also transferred dye much less efficiently at 24 h, in a manner dependent on PKC. However, keratinocyte migration to fill the scratch required early (within <6 h) gap junctional communication. Our evidence indicates that PKC-dependent phosphorylation of Cx43 at S368 creates dynamic communication compartments that can temporally and spatially regulate wound healing.

Hypoxia-Reoxygenation Inhibits Gap Junctional Communication in Cultured Human Umbilical Vein Endothelial Cells

Endothelium ◽  
2000 ◽  
Vol 7 (4) ◽  
pp. 279-286 ◽  
Author(s):  
Masamichi Nishida ◽  
Satoru Futami ◽  
Ikuo Morita ◽  
Kazuhiko Maekawa ◽  
Sci-Itsu Murota
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Umbilical Vein Endothelial Cells ◽  
Gap Junctional ◽  
Hypoxia Reoxygenation

scholarly journals Asymmetric patterns of gap junctional communication in developing chicken skin

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 85-96 ◽  
Author(s):  
F. Serras ◽  
S. Fraser ◽  
C.M. Chuong
Keyword(s):  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Coupling ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Chicken Skin ◽  
Preferential Pathways ◽  
Gap Junctional ◽  
Feather Development

To study the pattern of gap junctional communication in chicken skin and feather development, we injected Lucifer Yellow into single cells and monitored the transfer of the fluorescent dye through gap junctions. Dye coupling is present between cells of the epithelium as well as between cells of the mesoderm. However, dye transfer did not occur equally in all directions and showed several consistent patterns and asymmetries, including: (1) no dye coupling between mesoderm and epithelium, (2) partial restriction of dye coupling at the feather bud/interbud boundary during early feather bud development, (3) preferential distribution of Lucifer Yellow along the anteroposterior axis of the feather placode and (4) absence of dye coupling in some epithelial cells. These results suggest the presence of preferential pathways of communication that may play a role in the patterning of chicken skin.

VEGF transiently disrupts gap junctional communication in endothelial cells

2001 ◽  
Vol 114 (6) ◽  
pp. 1229-1235
Author(s):  
S. Suarez ◽  
K. Ballmer-Hofer
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Tyrosine Kinase ◽  
Map Kinases ◽  
Cell Junctions ◽  
Early Effect ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Vascular endothelial growth factor, VEGF, stimulates angiogenesis by directly acting on endothelial cells. The effects of VEGF are mediated by two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR) that are highly related to receptors of the platelet derived growth factor (PDGF) receptor family. We are interested in early signalling events downstream from VEGF receptors that affect blood vessel homeostasis. Endothelial cells form multiple types of cell-cell junctions that are required for cellular organization into complex networks. These junctions also regulate communication among adjacent cells. Stimulation by various growth factors such as epidermal growth factor (EGF) or PDGF has been shown to disrupt cell-cell junctions, consequently affecting cell-to-cell communication. We investigated gap junctional communication (GJC) by monitoring the transfer of a low molecular mass fluorescent tracer molecule between adjacent cells using immunofluorescence microscopy. VEGF maximally blocked GJC 15 minutes after growth factor administration. The cells resumed communication via gap junctions within 1–2 hours after treatment. This early effect of VEGF on communication correlated with changes in the phosphorylation state of one of the proteins involved in gap junction formation, connexin 43 (Cx43). The signalling mechanisms involved in this phenomenon depend on activation of VEGFR-2, impinge on a tyrosine kinase of the Src family and activate the Erk family of MAP kinases. The function of VEGF-mediated disruption of GJC might be to restrict an increase in endothelium permeability to the environment affected by local injury to blood vessels.

Gap junctions and fluid flow response in MC3T3-E1 cells

2001 ◽  
Vol 281 (6) ◽  
pp. C1917-C1925 ◽  
Author(s):  
M. M. Saunders ◽  
J. You ◽  
J. E. Trosko ◽  
H. Yamasaki ◽  
Z. Li ◽  
...  
Keyword(s):  
Fluid Flow ◽  
Gap Junctions ◽  
Bone Cell ◽  
Dominant Negative ◽  
Prostaglandin E ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Text Response ◽  
Gap Junctional

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca2+concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E2 (PGE2) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca2+ (Ca[Formula: see text]) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE2 but not to the Ca[Formula: see text] response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

2001 ◽  
Vol 280 (6) ◽  
pp. H2441-H2450 ◽  
Author(s):  
Andrew T. Chaytor ◽  
Patricia E. M. Martin ◽  
David H. Edwards ◽  
Tudor M. Griffith
Keyword(s):  
Gap Junctions ◽  
Hepatic Artery ◽  
Lucifer Yellow ◽  
Dye Transfer ◽  
Cell Coupling ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
A7r5 Cells ◽  
Gap Junctional

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26,43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 μM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 μM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

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