Na(+)/K(+)-ATPase as an energy transducing ion pump has been studied extensively since its discovery in 1957. Although early findings suggested a role for Na(+)/K(+)-ATPase in regulation of cell growth and expression of various genes, only in recent years the mechanisms through which this plasma membrane enzyme communicates with the nucleus have been studied. This research, carried out mostly on cardiac myocytes, shows that in addition to pumping ions, Na(+)/K+-ATPase interacts with neighboring membrane proteins and organized cytosolic cascades of signaling proteins to send messages to the intracellular organelles. The signaling pathways that are rapidly elicited by the interaction of ouabain with Na(+)/K(+)-ATPase, and are independent of changes in intracellular Na(+) and K(+) concentrations, include activation of Src kinase, transactivation of the epidermal growth factor receptor by Src, activation of Ras and p42/44 mitogen-activated protein kinases, and increased generation of reactive oxygen species by mitochondria. In cardiac myocytes, the resulting downstream events include the induction of some early response proto-oncogenes, activation of the transcription factors, activator protein-1 and nuclear factor kappa-B, regulation of a number of cardiac growth-related genes, and stimulation of protein synthesis and myocyte hypertrophy. For these downstream events, the induced reactive oxygen species and rise in intracellular Ca(2+) are essential second messengers. In cells other than cardiac myocytes, the proximal pathways linked to Na(+)/K(+)-ATPase through protein-protein interactions are similar to those reported in myocytes, but the downstream events and consequences may be significantly different. The likely extracellular physiological stimuli for the signal transducing function of Na+/K+-ATPase are the endogenous ouabain-like hormones, and changes in extracellular K+ concentration.

Nontoxic concentrations of ouabain, causing partial inhibition of the cardiac myocyte Na(+)/K(+)-ATPase, induce hypertrophy and several growth-related genes through signal pathways that include the activation of Ras and p42/44 mitogen-activated protein kinase (MAPK). The aim of this work was to examine the ouabain-induced events upstream of the Ras/MAPK cascade. Treatment of myocytes with genistein antagonized ouabain-induced activation of the MAPK, suggesting that protein tyrosine phosphorylation has a role. Tyrosine phosphorylation of several myocyte proteins was increased rapidly upon cell exposure to ouabain. Lowering of extracellular K(+) had a similar ouabain-like effect. Ouabain also increased protein tyrosine phosphorylation in A7r5, HeLa, and L929 cells. In cardiac myocytes and A7r5 cells, herbimycin A antagonized the ouabain-induced increase in protein tyrosine phosphorylation and MAPK activation. In both cell types, ouabain stimulated Src kinase activity, Src translocation to the Triton-insoluble fraction, Src association with the epidermal growth factor receptor, and the tyrosine phosphorylation of this receptor on site(s) other than its major autophosphorylation site, Tyr(1173). The findings suggest that (a) the ouabain-induced activation of Src and the Src-induced phosphorylation of the growth factor receptor provide the scaffolding for the recruitment of adaptor proteins and Ras and the activation of Ras/MAPK cascade; and (b) the activation of such pathways may be a common feature of the signal-transducing function of Na(+)/K(+)-ATPase in most cells.

We showed before that in cardiac myocytes partial inhibition of Na ؉ /K ؉ -ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca 2؉ -dependent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases. The aim of this work was to explore the roles of intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal ␣-actin and atrial natriuretic factor, repression of the gene of the ␣ 3 -subunit of Na ؉ /K ؉ -ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-B. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb؉ uptake through Na ؉ /K ؉ -ATPase and the resulting rise in intracellular Ca 2؉ were also not prevented by NAC. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation, and its effects on marker genes and protein synthesis were not affected by NAC. We conclude the following: (a) ROS are essential second messengers within some but not all signal pathways that are activated by the effect of ouabain on Na ؉ /K ؉ -ATPase; (b) the ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte.

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na

Exposure of neonatal rat cardiac myocytes to ouabain concentrations that caused partial inhibition of Na+/K+-ATPase but no loss of viability, increased c-fos and c-jun mRNAs and the transcription factor AP-1. The increased mRNAs were proportional to the extent of inhibition of Na+/K+-ATPase and the resulting rise in steady state intracellular Ca2+ concentration. The rapid and sustained increase of c-fos mRNA was shown to be due to increased transcriptional rate. Induction of c-fos by ouabain was prevented when either extracellular or intracellular Ca2+ was lowered and was attenuated by pretreatment of myocytes with a phorbol ester under conditions known to down-regulate protein kinase C. Exposure to ouabain for 24-48 h also increased total transcriptional activity and protein content of myocytes. The findings suggest that the same signal responsible for the positive inotropic action of ouabain, i.e. net influx of Ca2+ caused by partial inhibition of Na+/K+-ATPase, also initiates the rapid protein kinase C-dependent inductions of the early-response genes, the subsequent regulations of other cardiac genes by the resulting transcription factors, and stimulation of myocyte growth. Whether these hitherto unrecognized effects of cardiac glycosides are obtained in the intact heart and their relevance to the therapeutic uses of these drugs remain to be determined.

Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

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Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.