Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations

We hare shown previously that partial inhibition of the cardiac myocyte Na/K+-ATPase activates signal pathways that regulate myocyte growth and growt h-related genes and that increases in intracellular Ca2+ concentration ([Ca 2+](i)) and reactive oxygen species (ROS) are two essential second messenge rs 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, ouabai n caused no change in [Ca2+](i), but it increased ROS as it did when the ce lls 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 oua bain, was also independent of changes in [Ca2+](i) and [Na+](i). Ouabain-in duced generation of ROS in myocytes was antagonized by genistein, a dominan t negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochond rial origin for the Ras-dependent ROS generation. These findings, along wit h our previous data, indicate that increases in [Ca2+](i) and ROS in cardia c myocytes are induced by two parallel pathways initiated at the plasma mem brane: One being the ouabain-altered transient interactions of a fraction o f the Na+/K+-ATPase with neighboring proteins (Src, growth factor receptors , adaptor proteins, and Ras) leading to ROS generation, and the other, inhi bition of the transport function of another fraction of the Na+/K+-ATPase l eading to rise in [Ca2+](i). Evidently, the gene regulatory effects of ouab ain in cardiac myocytes require the downstream collaborations of ROS and [C a2+](i).