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Ouabain Binding to Na+,K+-ATPase Relaxes Cell Attachment and Sends a SpecificSignal (NACos) to the Nucleus

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Abstract.

In previous work we described a “P→A mechanism” that transduces occupancy of the pump (P) by ouabain into changes in phosphorylation, stimulation of mitogen-activated protein kinase (MAPK), and endocytosis of cell-cell- and cell-substrate-attaching molecules (A), thereby causing a release of the cell from the monolayer. In the present work we try to understand the mechanism of this effect; whether, in order to trigger the P→A mechanism, ouabain should block the pumping activity of Na+,K+-ATPase as pump, or whether it would suffice that the drug occupies this enzyme as a receptor. We assay a series of drugs known to act on the pump, such as ouabain, digoxin, digitoxin, palytoxin, oligomycin, strophanthidin, neothyoside-A, proscillaridin-A, etc. We gauge their ability to block the pump by measuring the K+ content in the cells, and their ability to detach the cells from the monolayer by determining the amount of protein remaining in the culturing well. None of the drugs tested was able to cause detachment without stopping the pump. Ouabain also enhances phosphorylation, yet pump inhibition and signal transduction do not seem to be intimately associated in a causal chain, but to occur simultaneously. To investigate the response of the site of cell attachment, we analyze the position of β-catenin by fluorescence confocal microscopy, and find that this adherent junction-associated molecule is sent to the nucleus, where it is known to act as a transcriptional cofactor.

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Acknowledgements

We thank Drs. G. Bianchi and Patrizia Ferrari from the Prassis Research Institute (Milano, Italy), who have generously provided the PST 2238 and PST 2744 that they synthesized; Dr. Rosalba Encarnación from the Marine Biological Department (University of Baja California, México) for providing neothyoside A, and Dr. Luz María del Razo for allowing us to use her atomic emission spectrometer to measure K, and helping us with the procedures. We also thank Mrs. E. del Oso and Y. de Lorenz for administrative help. This work was performed with the economic support of the National Research Council of México (CONACYT).

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  1. Department of Physiology, Biophysics and Neurosciences, Av., Center for Research & Advanced Studies, Instituto Politécnico Nacional 2508, México, D.F., 07300, Mexico

    R. G. Contreras, C. Flores-Maldonado, A. Lázaro, L. Shoshani, D. Flores-Benitez, I. Larré & M. Cereijido

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  1. R. G. Contreras
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  2. C. Flores-Maldonado
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  3. A. Lázaro
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  4. L. Shoshani
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  7. M. Cereijido
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Contreras, R.G., Flores-Maldonado, C., Lázaro, A. et al. Ouabain Binding to Na+,K+-ATPase Relaxes Cell Attachment and Sends a SpecificSignal (NACos) to the Nucleus. J Membrane Biol 198, 147–158 (2004). https://doi.org/10.1007/s00232-004-0670-2

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