scholarly journals Ouabain Enhances Gap Junctional Intercellular Communication by Inducing Paracrine Secretion of Prostaglandin E2

2021 ◽  
Vol 22 (12) ◽  
pp. 6244
Author(s):  
Alejandro Ogazon del Toro ◽  
Lidia Jimenez ◽  
Mauricio Serrano Rubi ◽  
Marcelino Cereijido ◽  
Arturo Ponce
Keyword(s):  
Prostaglandin E2 ◽  
Intercellular Communication ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Direct Stimulation ◽  
Paracrine Secretion ◽  
Pre Treatment ◽  
Epithelial Physiology ◽  
Gap Junctional ◽  
Mdck I

Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM’s ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.

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.

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.

Loss of gap junctional intercellular communication in rat lung epithelial cells exposed to carbon or silica-based nanoparticles

2010 ◽  
Vol 391 (11) ◽  
Author(s):  
Niloofar Ale-Agha ◽  
Catrin Albrecht ◽  
Lars-Oliver Klotz
Keyword(s):  
Epithelial Cells ◽  
Intercellular Communication ◽  
Connexin 43 ◽  
Lung Epithelial Cells ◽  
Rat Lung ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Junction Protein ◽  
Lung Epithelial ◽  
Gap Junctional

Abstract The aim of this study was to investigate whether fine and ultrafine carbon black (fC and ufC), and fine and ultrafine silica (fS, ufS) particles affect gap junctional intercellular communication (GJIC) in rat lung epithelial cells. Exposure of cells to subcytotoxic doses of ufC, fS and ufS resulted in a 63%, 59% and 77% reduction of GJIC, respectively, as determined in a dye transfer assay. In contrast to ufC, fC did not significantly alter GJIC. Changes in subcellular localization of the major gap junction protein in RLE cells, connexin-43 (Cx43), and of β-catenin were observed in cells exposed to ufC, fS or ufS. The loss of GJIC was counteracted by N-acetyl cysteine and was largely prevented by specific inhibitors of epidermal growth factor receptor-dependent signaling, pointing to the crucial role of two known major mediators of nanoparticle action, namely reactive oxygen species and membrane-receptor signaling, in particle-induced modulation of GJIC.

scholarly journals Prostaglandin E2 Enhances Gap Junctional Intercellular Communication in Clonal Epithelial Cells

2021 ◽  
Vol 22 (11) ◽  
pp. 5813
Author(s):  
Alejandro Ogazon del Toro ◽  
Lidia Jimenez ◽  
Mauricio Serrano Rubi ◽  
Aida Castillo ◽  
Lorena Hinojosa ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Prostaglandin E2 ◽  
Protein Kinase A ◽  
Intercellular Communication ◽  
Mdck Cells ◽  
Lucifer Yellow ◽  
Gap Junctional Intercellular Communication ◽  
Gap Junctional ◽  
Kinase A

Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.

scholarly journals Ouabain Promotes Gap Junctional Intercellular Communication in Cancer Cells

2020 ◽  
Vol 22 (1) ◽  
pp. 358
Author(s):  
Mauricio Serrano-Rubi ◽  
Lidia Jimenez ◽  
Jacqueline Martinez-Rendon ◽  
Marcelino Cereijido ◽  
Arturo Ponce
Keyword(s):  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Intercellular Communication ◽  
Cancer Cell Lines ◽  
Molecular Structures ◽  
Dye Transfer ◽  
Gap Junctional Intercellular Communication ◽  
Therapeutic Properties ◽  
Gap Junctional

Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.

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