Partial inhibition of cardiac Na/K-ATPase by digitalis drugs such as ouabain is the initial event leading to positive inotropy in the heart. We showed recently that exposure of rat cardiac myocytes to ouabain concentrations that produce positive inotropy, but no overt toxicity, caused inductions of some early response genes and hypertrophy of these myocytes. The aim of this work was to determine if ouabain also affects the expressions of certain late response genes that are regulated by other hypertrophic stimuli. Non-toxic concentrations of ouabain (5-100 microM) increased mRNAs of skeletal alpha-actin, atrial natriuretic factor, myosin light chain 2, and transforming growth factor beta: indicating that ouabain's effects on these marker genes are similar to those of hypertrophic stimuli that mimic the effects of pressure overload. Expression of skeletal alpha-actin was more sensitive to ouabain than that of atrial natriuretic factor, suggesting significant differences in the ouabain-specific pathways of the induction of these fetal genes. The effects of ouabain on skeletal alpha-actin gene were transcriptional, and required an increase in net influx of extracellular Ca2+. Protein kinase C and Ca(2+)-calmodulin kinases, but not protein kinase A, were involved in the signal pathways leading to the induction of skeletal alpha-actin gene. These data and our prior findings indicate that an increase in net influx of Ca2+ through partial inhibition of Na/K-ATPase initiates protein kinase-dependent pathways resulting in alterations in cardiac growth and expressions of both early and late response genes.