Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations.
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 Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](i) and ROS. When myocytes were in a Ca(2+)-free medium, ouabain caused no change in [Ca(2+)](i), but it increased ROS as it did when the cells were in a Ca(2+)-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 Ca(2+)-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 [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i). Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca(2+)](i).
Liu
J
J
Departments of Pharmacology and Medicine, Medical College of Ohio, Toledo, Ohio 43614, USA.
Tian
J
J
Haas
M
M
Shapiro
J I
JI
Askari
A
A
Xie
Z
Z
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D019833:Genistein / Q000494:pharmacology; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008954:Models, Biological; D008985:Monensin / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D017382:Reactive Oxygen Species; D010042:Ouabain; D008985:Monensin; D012964:Sodium; D019833:Genistein; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium