The Bioactive Phenolic Agents Diaryl Ether CVB2-61 and Diarylheptanoid CVB4-57 as Connexin Hemichannel Blockers.

Inflammation mediators enhance the activity of connexin (Cx) hemichannels, especially in the epithelial and endothelial tissues. As potential release routes for injury signals, such as (oligo)nucleotides, Cx hemichannels may contribute to long-lasting inflammation. Specific inhibition of Cx hemichannels may therefore be a mode of prevention and treatment of long-lasting, chronic sterile inflammation. The activity of Cx hemichannels was analysed in N2A and HeLa cells transfected with human Cx26 and Cx46 as well as in Calu-3 cells, using dye uptake as functional assay. Moreover, the possible impacts of the bioactive phenolic agents CVB2-61 and CVB4-57 on the barrier function of epithelial cells was analysed using Calu-3 cells. Both agents inhibited the dye uptake in N2A cells expressing Cx26 (>5 µM) and Cx46 (>20 µM). In Calu-3 cells, CVB2-61 and CVB4-57 reversibly inhibited the dye uptake at concentrations as low as 5 µM, without affecting the gap junction communication and barrier function, even at concentrations of 20 µM. While CVB2-61 or CVB4-57 maintained a reduced dye uptake in Calu-3 cells, an enhancement of the dye uptake in response to the stimulation of adenosine signalling was still observed after removal of the agents. The report shows that CVB2-61 and CVB4-57 reversibly block Cx hemichannels. Deciphering the mechanisms of the interactions of these agents with Cx hemichannels could allow further development of phenolic compounds to target Cx hemichannels for better and safer treatment of pathologies that involve Cx hemichannels.

Stimulation of the A2B Adenosine Receptor Subtype Enhances Connexin26 Hemichannel Activity in Small Airway Epithelial Cells.

BACKGROUND/AIMS: Adenosine release and connexin (Cx) hemichannel activity are enhanced in the respiratory epithelium during pathophysiological events such as inflammation. We analysed the interplay between Cx channels and adenosine signalling in human respiratory airway epithelium using the Calu-3 cell line as a model. METHODS: The Cx hemichannel activity in Calu-3 cells was evaluated by dye uptake assays. The expressed Cx isoforms and adenosine receptor subtypes were identified by PCR and western blot analysis. Pharmacological and molecular biological experiments were performed to analyse the involvement of the different adenosine receptor subtypes, the induced signalling pathways and the contribution of specific Cx isoforms to the hemichannel activity. RESULTS: The adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the dye uptake rate in Calu-3 cells. The pannexon and Cx hemichannel inhibitor carbenoxolone (CBX) did not supress the dye uptake at pannexin-specific concentrations (100 µM). High CBX concentrations or the inhibitor La3+, both effective on Cx hemichannels, were needed to inhibit the dye uptake. The NECA-related increase of dye uptake depended on enhanced cAMP synthesis and subsequent activation of the protein kinase A (PKA) as shown by quantification of cAMP levels and pharmacological inhibition of the adenylyl cyclase and the PKA. Further pharmacological inhibition as well as knockdown experiments with specific siRNA showed that the A2B adenosine receptor was the subtype mainly responsible for the increased dye uptake. The NECA-related increase of the dye uptake rate correlated with a decrease of Cx43 mRNA and an increase of Cx26 mRNA content in the cells as well as Cx26 protein synthesis and was inhibited by Cx26 knockdown using Cx26 siRNA. Of note, a siRNA-induced knockdown of Cx43 increased the content of Cx26 mRNA and correspondingly the dye uptake rate. CONCLUSION: The Calu-3 cell model shows that stimulation of the A2B adenosine receptor subtype activates synthesis of cAMP. cAMP activates PKA and induces thereby an increase in Cx26 and a decrease in Cx43 mRNA levels. As a result, the synthesis of Cx26 is reinforced, leading to an enhanced Cx hemichannel activity. The report identifies a mechanism that integrates adenosine release and Cx hemichannel activity and shows how adenosine signalling and Cx channels may act together to promote persistent inflammation, which is observed in several chronic diseases of the respiratory airway.

Tumour-treating fields (TTFields): Investigations on the mechanism of action by electromagnetic exposure of cells in telophase/cytokinesis.

Tumour-treating fields (TTFields) use alternating electric fields which interfere with dividing cells, thereby reducing tumour growth. Previous reports suggest that electrical forces on cell structure proteins interfered with the chromosome separation during mitosis and induced apoptosis. In the present report we evaluate electromagnetic exposure of cells in telophase/cytokinesis in order to further analyse the mechanism of action on cells. We performed numerical electromagnetic simulations to analyse the field distribution in a cell during different mitotic phases. Based thereon, we developed an electric lumped element model of the mitotic cell. Both the electromagnetic simulation and the lumped element model predict a local increase of the specific absorption rate (SAR) as a measure of the electromagnetically induced power absorption density at the mitotic furrow which may help to explain the anti-proliferative effect. In accordance with other reports, cell culture experiments confirmed that TTFields reduce the proliferation of different glioma cell lines in a field strength- and frequency-dependent manner. Furthermore, we found an additional dependence on the commutation time of the electrical fields. The report gives new insights into TTFields' anti-proliferative effect on tumours, which could help to improve future TTFields application systems.