The effect of cardiac glycosides to increase cardiac inotropy by altering Ca2+ cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca2+-ATPase function. Rat cardiac myocytes treated with 50 mu M ouabain demonstrated substantial increases in systolic and diastolic Ca2+ concentrations. The recovery time constant for the Ca2+ transient, tau(Ca2+), was significantly prolonged by ouabain. Exposure to 10 mu M H2O2, which causes an increase in intracellular reactive oxygen species similar to that of 50 mu M ouabain, caused a similar increase in tau(Ca2+). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea ( 50 mg/ml) both prevented the increases in tau(Ca2+) as well as the changes in systolic or diastolic Ca2+ concentrations. We also observed that 50 mu M ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca2+-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca2+-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca2+-ATPase structure and function that may, in turn, produce some of the associated changes in Ca2+ cycling and physiological function.