Ouabain Interaction with Cardiac Na+/K+-ATPase Initiates Signal Cascades Independent of Changes in Intracellular Na+ and Ca2+ Concentrations

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Abstract
We have shown previously that partial inhibition of the cardiac myocyte Na+/K+-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca2+ concentration ([Ca2+] i ) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca2+] i and ROS. When myocytes were in a Ca2+-free medium, ouabain caused no change in [Ca2+] i , but it increased ROS as it did when the cells were in a Ca2+-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na+] i . Exposure of myocytes in Ca2+-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca2+] i and [Na+] i . Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca2+] i and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na+/K+-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na+/K+-ATPase leading to rise in [Ca2+] i . Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca2+] i .