Collective cell migration is important for many physiological and pathological processes. Ouabain, a cardiotonic steroid and specific ligand to Na,K-ATPase, has been reported to modulate collective cell migration, inhibiting it in cancer cells, and increasing it in normal cells. However, it is not clear what the mechanism is behind this difference in response. It is well-documented that ouabain blocks the pump function of Na,K-ATPase. At low concentrations this activates on the cellular level a Ca2+ mediated signaling chain with multiple downstream effects, including adhesion and migration pathways. At higher concentrations the inhibition of Na+ and K+ transport dominates the effect on the cellular level. We hypothesize that the difference in migration response of cancer and normal cells to ouabain is due to a difference in the Ca2+ signaling response. We further hypothesize that the spread and amplification of Ca2+signals between cells via gap junctions is upregulated by ouabain. To test the hypothesis, we made scratch assays in cultures of MDCK cells and studied migration rate and Ca2+signaling. 10 nM ouabain increases the migration rate, an effect that is inhibited by 2-APB, that blocks the IP3R Ca2+ release from ER. The effect on migration is also blocked by KN-93 that inhibits Ca2+ activated CAMKII. Interestingly, ouabain at 200 nM concentration decreased the migration rate compared to untreated cells. To analyze the strength of intercellular communication we analyzed the spread and synchronicity of spontaneous Ca2+ signals between neighboring cells. For this we used MDCK cells stable transfected with the Ca2+ sensitive fluorescent protein GCamp6f. Cells were treated with vehicle, ouabain 10 nM, or heptanol (blocks gap junctions). Timelapse Ca2+ imaging for 10-30 minutes was done. Machine learning based image analysis with Cellpose was then used to segment cells, followed by cross correlation of the integrated Ca2+signals from neighboring cells. Despite findings from previous phosphoproteomic studies indicating that there should be an upregulation of gap junctions, no conclusive differences in intercellular signaling could be identified for the different treatments. Finally, we made high resolution imaging of cells immunolabeled for vinculin and FAK, two proteins that are involved in migration and adhesion. We found ouabain correlated effects on the localization of vinculin and FAK with a polarized and concentrated localization after high dose ouabain, compared to a diffuse and unpolarized localization after low dose ouabain. Taken together we suggest that low dose ouabain via Na,K-ATPase triggers a Ca2+ signal that destabilize cell adhesions and that this is an important mechanism contributing to the observed upregulation of cell migration. Marta and Gunnar V Philipson's foundation, and VR 2020-05347 (Swedish Research Council). 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.