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Description:
By allowing a direct exchange of ions and metabolites between cells, gap junctions participate in the formation of physiological units in tissues. Gap junctions in endothelial cells are essential for maintaining vascular functions. Adenosine is a ubiquitous extracellular signalling molecule that can evoke cellular responses in large tissue areas by binding in a paracrine manner to its receptors. Adenosine receptor-dependent signalling mechanisms regulate several vascular functions, for example vasodilation or endothelial barrier properties. Since gap junctions are important for various tissue functions, it is essential to include the gap junction regulation into general signalling mechanisms within tissues. Therefore, the regulation of gap junction coupling by adenosine receptor signalling was analysed in the presented work. Activation of the adenosine receptor subtype A2B significantly increased the gap junction coupling and the amount of connexin43 gap junction plaques in microvascular endothelial hCMEC/D3 cells via activation of cyclic nucleotide-gated (CNG) channels. On functional level the regulation of gap junctions upon adenosine receptor activation and especially the involvement of CNG channels is as yet a disregarded signalling link and could provide new insights for example into the regulation of inflammatory conditions. Analysis of gap junction coupling was performed with scrape loading/dye transfer assays. To improve this technique a gold nanoparticle-mediated laser perforation/dye transfer (GNOME LP/DT) method was established for a non-invasive, cell-friendly analysis of gap junction-dependent cell coupling. The GNOME LP/DT method enabled the analysis of gap junction coupling with similar results as scrape loading/dye transfer assays and was more reproducible. Additionally, the GNOME LP/DT method was successfully applied to sensitive cells and in complex cell culture systems, for example three-dimensional cell culture or co-culture of blood-brain barrier cells in transwell inserts. Applying the ...