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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 ...