Signaling switches maintain intercellular communication in the vascular endothelium

The endothelium is a sophisticated coordination center that controls a wide range of vascular functions, which include the regulation of blood pressure and blood flow via changes in vascular contractility. To coordinate vascular function, cell communication and interactions are required for tissue level responses to emerge. A significant form of cell-cell communication occurs by the propagation of Ca 2+ signals between cells (‘Ca 2+ waves’). The objective of our study was to tease apart the mechanisms driving these intercellular Ca 2+ waves in the vascular endothelium. We hypothesised that diffusion of free Ca 2+ would drive endothelial Ca 2+ wave propagation and effect vessel diameter changes. To investigate the mechanisms, intact mesenteric arteries from male Sprague Dawley rats were mounted in an en face preparation, stained with the Ca 2+ indicator Cal-520/AM and were imaged on specialised epifluorescence microscopes. Intercellular endothelial signaling was manipulated using caged compounds in concert with precise spatial and temporal control of UV illumination, as well as global or localized drug application. We show that multiple mechanisms maintain communication so that Ca 2+ wave propagation occurs irrespective of the status of cell connectivity. Contrary to our hypothesis, regenerative IP 3 -induced IP 3 production transmits Ca 2+ signals between adjoining cells and explains the propagated vasodilation that underlies the increased blood flow accompanying tissue activity. By controlling the production of IP 3 , the inositide is itself suffcient to evoke a regenerative phospholipase C-dependent Ca 2+ wave across coupled cells. None of gap junctions, Ca 2+ diffusion nor the release of extracellular messengers are required to support this type of intercellular Ca 2+ signaling. Surprisingly, when a discontinuity between cells was created using a fire-polished pipette tip, signal was propagated to the other side of the discontinuity and was able to drive propagated vasodilation. Experiments using counter flow and localised perfusion in the absence and presence of P2Y inhibitors revealed that it is ATP released as a diffusible extracellular messenger that transmits Ca 2+ signals across endothelial discontinuities. These results show that signaling switches underlie endothelial cell-cell signal transmission, and reveal how communication is maintained in the face of endothelial damage. The findings provide a new framework for understanding Ca 2+ wave propagation and cell-cell signaling in the endothelium. This work was funded by the British Heart Foundation (RG/F/20/110007; PG/20/9/34859 ). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.