Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

2006 ◽  
Vol 290 (5) ◽  
pp. H2015-H2023 ◽  
Author(s):  
Eno Essien Ebong ◽  
Sanghee Kim ◽  
Natacha DePaola
Keyword(s):  
Endothelial Cells ◽  
Gap Junctions ◽  
Intercellular Communication ◽  
Flow Chamber ◽  
Functional Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Dynamic Regulation ◽  
Gap Junctional

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.

scholarly journals High-throughput measurement of gap junctional intercellular communication

2014 ◽  
Vol 306 (12) ◽  
pp. H1708-H1713 ◽  
Author(s):  
Jun Liu ◽  
Vinayakumar Siragam ◽  
Jun Chen ◽  
Michael D. Fridman ◽  
Robert M. Hamilton ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
High Throughput ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Cell Injection ◽  
Operation Speed ◽  
Gap Junctional

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Abstract 335: Carbon Nanoparticles Cause Loss of Gap Junctional Intercellular Communication and Nuclear Translocation of ß-Catenin in Endothelial Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Niloofar Ale-Agha ◽  
Klaus Unfried ◽  
Joachim Altschmied ◽  
Judith Haendeler
Keyword(s):  
Endothelial Cells ◽  
Intercellular Communication ◽  
Carbon Nanoparticles ◽  
Connexin 43 ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Cell Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junctional

Introduction: Environmental factors like industrial or consumer derived pollution are known to increase the risk for cardiovascular diseases. Previous studies demonstrated that senescent endothelial cells contribute to the onset of atherosclerosis. We have recently shown that non-toxic and non-inflammatory concentrations of carbon nanoparticles (CNP), a major component of industrial pollution, induce senescence in human primary endothelial cells (EC). However, the underlying molecular mechanisms leading to senescent EC are far from understood. Gap junctional intercellular communication (GJIC), which depends on Connexin 43 (Cx43) and β-catenin, is important for endothelial function and seems to be dysregulated during aging. Phosphorylation of Cx43 on serine 368 reduces the trafficking of Cx43 to the plasma membrane and assembly of Cx43 into gap junctional structures. Objective: The aim of this study was to investigate whether CNP affect GJIC and β-catenin in EC. Material and Methods: EC were treated with CNP at a measured size of 10 - 34 nm and GJIC was measured by dye transfer assay. The localization of Cx43 and β-catenin was assessed by immunofluorescence and phosphorylation and protein levels of Cx43 were by immunoblots. Results: Two weeks incubation with CNP concentrations, which we are exposed to everyday, elevated the number of senescent EC. This was determined by increased staining for senescence-associated beta-galactosidase and the cell cycle inhibitor p21 - two established markers of senescence. The same concentrations of CNP resulted in an enhanced phosphorylation of Cx43 on serine 368. Moreover, CNP induced internalization of Cx43 and led to nuclear translocation of the Cx43 interacting protein β-catenin. This resulted in reduced GJIC, which was shown by decreased dye transfer. Thus, CNP-induced senescence is accompanied by loss of cell-cell communication. Conclusion: These data indicate for the first time that Cx43 and β-catenin, which are essential for endothelial functionality, are affected by CNP and that loss of GJIC is involved in CNP-induced senescence. Overexpression studies will investigate whether a causal link exists between CNP-induced senescence and loss of GJIC in primary EC.

scholarly journals Low pH enhances connexin32 degradation in the pancreatic acinar cell

2014 ◽  
Vol 307 (1) ◽  
pp. G24-G32 ◽  
Author(s):  
Anamika M. Reed ◽  
Thomas Kolodecik ◽  
Sohail Z. Husain ◽  
Fred S. Gorelick
Keyword(s):  
Gap Junctions ◽  
Acinar Cell ◽  
Intercellular Communication ◽  
Pancreatic Acinar Cell ◽  
Low Ph ◽  
Extracellular Ph ◽  
Gap Junctional Intercellular Communication ◽  
Junction Protein ◽  
Clinical Conditions ◽  
Gap Junctional

Decreased extracellular pH is observed in a number of clinical conditions and can sensitize to the development and worsen the severity of acute pancreatitis. Because intercellular communication through gap junctions is pH-sensitive and modulates pancreatitis responses, we evaluated the effects of low pH on gap junctions in the rat pancreatic acinar cell. Decreasing extracellular pH from 7.4 to 7.0 significantly inhibited gap junctional intracellular communication. Acidic pH also significantly reduced levels of connexin32, the predominant gap junction protein in acinar cells, and altered its localization. Increased degradation through the proteasomal, lysosomal, and autophagic pathways mediated the decrease in connexin32 under low-pH conditions. These findings provide the first evidence that low extracellular pH can regulate gap junctional intercellular communication by enhancing connexin degradation.

Mutagenic and epigenetic mechanisms of zearalenone: prevention by Vitamin E

2008 ◽  
Vol 1 (3) ◽  
pp. 369-374 ◽  
Author(s):  
Z. Ouanes-Ben Othmen ◽  
S. Essefi ◽  
H. Bacha
Keyword(s):  
Vitamin E ◽  
Intercellular Communication ◽  
Chromosome Aberrations ◽  
Vero Cells ◽  
Antioxidant Vitamin ◽  
Epigenetic Mechanisms ◽  
Gap Junctional Intercellular Communication ◽  
Epigenetic Effects ◽  
Gap Junctional

It has been suggested that zearalenone, a non-steroidal estrogenic mycotoxin produced by Fusarium graminearium, causes DNA damage. However, the mutagenic properties of this toxin are controversial. The purpose of this study was to investigate both genotoxic and epigenetic effects of zearalenone in vitro. The effects of zearalenone on unscheduled DNA synthesis (UDS), induction of chromosome aberrations and inhibition of gap junctional intercellular communication were determined using Vero cells. The results show that in Vero cells, zearalenone treatment caused a concentration-dependent increase in UDS, induced chromosome aberrations and inhibited gap junctional intercellular communication. All of these effects were either prevented or reduced by co-treatment with the antioxidant vitamin E. The results support the hypothesis that in Vero cells zearalenone-induced oxidative stress is involved in and precedes all of the studied effects.

scholarly journals Controlling cell–cell interactions using surface acoustic waves

2014 ◽  
Vol 112 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Feng Guo ◽  
Peng Li ◽  
Jarrod B. French ◽  
Zhangming Mao ◽  
Hong Zhao ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Acoustic Waves ◽  
Cancer Metastasis ◽  
Surface Acoustic Waves ◽  
Spatial Arrangement ◽  
Gap Junctional Intercellular Communication ◽  
Wave Technique ◽  
Immunological Reactions ◽  
Gap Junctional

The interactions between pairs of cells and within multicellular assemblies are critical to many biological processes such as intercellular communication, tissue and organ formation, immunological reactions, and cancer metastasis. The ability to precisely control the position of cells relative to one another and within larger cellular assemblies will enable the investigation and characterization of phenomena not currently accessible by conventional in vitro methods. We present a versatile surface acoustic wave technique that is capable of controlling the intercellular distance and spatial arrangement of cells with micrometer level resolution. This technique is, to our knowledge, among the first of its kind to marry high precision and high throughput into a single extremely versatile and wholly biocompatible technology. We demonstrated the capabilities of the system to precisely control intercellular distance, assemble cells with defined geometries, maintain cellular assemblies in suspension, and translate these suspended assemblies to adherent states, all in a contactless, biocompatible manner. As an example of the power of this system, this technology was used to quantitatively investigate the gap junctional intercellular communication in several homotypic and heterotypic populations by visualizing the transfer of fluorescent dye between cells.

Transgenic Disruption of Gap Junctional Intercellular Communication Enhances Early but Not Late Stage Hepatocarcinogenesis in the Rat

2005 ◽  
Vol 33 (6) ◽  
pp. 695-701 ◽  
Author(s):  
Naomi Hokaiwado ◽  
Makoto Asamoto ◽  
Kumiko Ogawa ◽  
Tomoyuki Shirai
Keyword(s):  
Intercellular Communication ◽  
Early Stage ◽  
Intraperitoneal Administration ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Connexin 32 ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Transgenic Rats ◽  
Gap Junctional

Much experimental evidence supports the conclusion that loss of gap junctional intercellular communication (GJIC) contributes to carcinogenesis. Transgenic rats featuring a dominant negative mutant of the connexin 32 gene under albumin promoter control (Cx32ΔTg-High and Cx32ΔTg-Low lines, respectively with high and low copy numbers of the transgene) have disrupted GJIC, as demonstrated by scrape dye-transfer assay in vivo as previous report by Asamoto et al. (2004) . In the present study, we investigated the susceptibility of these transgenic rats to a single intraperitoneal administration of diethylnitrosamine (DEN), and found a significant increase in preneoplastic glutathione S-transferase placental form (GST-P) positive lesions in the livers of Cx32ΔTg-High but not Cx32ΔTg-Low rats. However, incidences of adenomas and hepatocellular carcinomas were not elevated at the end of the experiment (52 weeks). In addition, we investigated the promotional effect of phenobarbital (PB) on Cx32ΔTg-High rats pretreated with DEN and found enhanced formation of GST-P positive lesions, in contrast to the lack of promoting effects reported for Cx32 deficient mice. The results indicate that although both high and low expression of the dominant negative connexin 32 mutant gene in our rats is able to inhibit gap junctional capacity, only high expression is effective at enhancing susceptibility to early stage DEN-induced liver carcinogenesis.

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