Exposure of cultured rat cardiac myocytes to ouabain is known to cause the interaction of Na+/K+-ATPase with adjacent proteins, leading to activation of multiple signal transduction pathways, regulation of growth-related genes, and hypertrophy. The aim of this work was to determine if the proximal signaling events identified in cultured myocytes also occur in isolated intact hearts of rat and guinea pig in response to positive inotropic doses of ouabain. Langendorff rat heart preparations were exposed to 50 microM ouabain to produce positive inotropy without toxicity, and assayed for Src kinase, protein kinase C, and extracellular signal-regulated kinases 1 and 2 (ERK(1/2)). These kinases were rapidly activated by ouabain as in cultured cells. In isolated guinea pig hearts, 1 microM ouabain caused ERK(1/2) activation comparable to the effect of 50 microM ouabain in rat heart and consistent with the higher ouabain sensitivity of the contractility of guinea pig heart. These data show that the proximal ouabain-induced signal pathways previously noted in cultured cells are not artifacts of dispersion/culturing of myocytes, and are not the peculiar properties of the rat heart with its relatively low ouabain sensitivity. They also suggest that treatment with positive inotropic doses of cardiac glycosides is likely to be associated with changes in the cardiac phenotype.