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

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Here we show that ouabain-induced cell growth regulation is intrinsically coupled to changes in the cellular amount of Na/KATPase via the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. Ouabain increases the endocytosis and degradation of Na/K-ATPase in LLC-PK1, human breast (BT20), and prostate (DU145) cancer cells. However, ouabain stimulates the PI3K/Akt/mTOR pathway and consequently up-regulates the expression of Na/K-ATPase in LLC-PK1 but not BT20 and DU145 cells. This up-regulation is sufficient to replete the plasma membrane pool of Na/K-ATPase and to stimulate cell proliferation in LLC-PK1 cells. On the other hand, ouabain causes a gradual depletion of Na/K-ATPase and an increased expression of cell cycle inhibitor p21 cip , which consequently inhibits cell proliferation in BT20 and DU145 cells. Consistently, we observe that small interfering RNA-mediated knockdown of Na/K-ATPase is sufficient to induce the expression of p21 cip and slow the proliferation of LLC-PK1 cells. Moreover, this knockdown converts the growth stimulatory effect of ouabain to growth inhibition in LLC-PK1 cells. Mechanistically, both Src and caveolin-1 are required for ouabaininduced activation of Akt and up-regulation of Na/K-ATPase. Furthermore, inhibition of the PI3K/Akt/mTOR pathway by rapamycin completely blocks ouabain-induced expression of Na/K-ATPase and converts ouabain-induced growth stimulation to growth inhibition in LLC-PK1 cells. Taken together, we conclude that changes in the expression of Na/K-ATPase dictate the growth regulatory effects of ouabain on cells.The Na/K-ATPase, a member of P-type ATPase family, was discovered as an energy transducing ion pump. It transports Na ϩ and K ϩ across the cell membrane and maintains ion homeostasis in animal cells (1, 2). Recent studies indicate that the Na/K-ATPase is also an important receptor that can transduce ligand binding into the activation of protein kinase cascades (3). Specifically, the Na/K-ATPase interacts with Src, which provides at least two important cellular regulations (4, 5). First, association with Na/K-ATPase keeps Src in an inactive state. Thus, the Na/K-ATPase serves as a native negative Src regulator (4). Second, this interaction forms a functional receptor complex for cardiotonic steroids (CTS) 3 (3), a group of well characterized ligands of the Na/K-ATPase. Cardiotonic steroids include cardenolides (e.g. ouabain) and bufadienolides (e.g. marinobufagenin) (6). Although CTS are known cardiac drugs, some of them have now been identified as endogenous steroid hormones (6 -8). Binding of CTS to the receptor complex activates the Na/K-ATPase-associated Src. Subsequently, the activated Src transactivates other tyrosine kinases, and together they recruit and further phosphorylate multiple membrane and soluble proteins, which results in the activation of protein kinase cascades and the generation of second messengers (3, 4, 6). Ultimately, this chain of signaling events would alter cellular functions and cell growth in a cell-...

We have shown before that Na ؉ /K ؉ -ATPase acts as a signal transducer, through protein-protein interactions, in addition to being an ion pump. Interaction of ouabain with the enzyme of the intact cells causes activation of Src, transactivation of EGFR, and activation of the Ras/ ERK1/2 cascade. To determine the role of protein kinase C (PKC) in this pathway, neonatal rat cardiac myocytes were exposed to ouabain and assayed for translocation/ activation of PKC from cytosolic to particulate fractions. Ouabain caused rapid and sustained stimulation of this translocation, evidenced by the assay of Ca 2؉ -dependent and Ca 2؉ -independent PKC activities and by the immunoblot analysis of the ␣, ␦, and ⑀ isoforms of PKC. Dose-dependent stimulation of PKC translocation by ouabain (1-100 M) was accompanied by no more than 50% inhibition of Na ؉ /K ؉ -ATPase and doubling of [Ca 2؉ ] i , changes that do not affect myocyte viability and are known to be associated with positive inotropic, but not toxic, effects of ouabain in rat cardiac ventricles. Ouabain-induced activation of ERK1/2 was blocked by PKC inhibitors calphostin C and chelerythrine. An inhibitor of phosphoinositide turnover in myocytes also antagonized ouabain-induced PKC translocation and ERK1/2 activation. These and previous findings indicate that ouabain-induced activation of PKC and Ras, each linked to Na ؉ /K ؉ -ATPase through Src/EGFR, are both required for the activation of ERK1/2. Ouabain-induced PKC translocation and ERK1/2 activation were dependent on the presence of Ca 2؉ in the medium, suggesting that the signal-transducing and ion-pumping functions of Na ؉ /K ؉ -ATPase cooperate in activation of these protein kinases and the resulting regulation of contractility and growth of the cardiac myocyte.

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.

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Cultured rat cardiac myocytes and A7r5 cells were transfected with an adenoviral vector used earlier for in vivo expression of functional alpha(2)-isoform of the catalytic subunit of rat Na(+)-K(+)-ATPase. Expressions of truncated forms of alpha(2), but little or no intact alpha(2), were detected, suggesting the rapid degradation of alpha(2) in these cultured cells. In neonatal myocytes normally containing the alpha(1)- and the alpha(3)-isoforms, expression of the alpha(2)-fragment led to 1) a significant decrease in the level of endogenous alpha(1)-protein and a modest decrease in alpha(3)-protein, 2) decreases in mRNAs of alpha(1) and alpha(3), 3) decrease in Na(+)-K(+)-ATPase function measured as ouabain-sensitive Rb(+) uptake, 4) increase in intracellular Ca(2+) concentration similar to that induced by ouabain, and 5) eventual loss of cell viability. These findings indicate that the alpha(2)-fragment downregulates endogenous Na(+)-K(+)- ATPase most likely by dominant negative interference either with folding and/or assembly of the predominant housekeeping alpha(1)-isoform or with signal transducing function of the enzyme. Demonstration of rise in intracellular Ca(2+) resulting from alpha(1)-downregulation 1) does not support the previously suggested special roles of less abundant alpha(2)- and alpha(3)-isoforms in the regulation of cardiac Ca(2+), 2) lends indirect support to proposals that observed decrease in total Na(+)-K(+)-ATPase of the failing heart may be a mechanism to compensate for impaired cardiac contractility, and 3) suggests the potential therapeutic utility of dominant negative inhibition of Na(+)-K(+)-ATPase.