We have shown previously that partial inhibition of the cardiac myocyte Na⁺/K⁺-ATPase activates signal pathways that regulate myocyte growth and growth-related genes and that increases in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) and reactive oxygen species (ROS) are two essential second messengers within these pathways. The aim of this work was to explore the relation between [Ca²⁺] _i and ROS. When myocytes were in a Ca²⁺-free medium, ouabain caused no change in [Ca²⁺] _i , but it increased ROS as it did when the cells were in a Ca²⁺-containing medium. Ouabain-induced increase in ROS also occurred under conditions where there was little or no change in [Na⁺] _i . Exposure of myocytes in Ca²⁺-free medium to monensin did not increase ROS. Increase in protein tyrosine phosphorylation, an early event induced by ouabain, was also independent of changes in [Ca²⁺] _i and [Na⁺] _i . Ouabain-induced generation of ROS in myocytes was antagonized by genistein, a dominant negative Ras, and myxothiazol/diphenyleneiodonium, indicating a mitochondrial origin for the Ras-dependent ROS generation. These findings, along with our previous data, indicate that increases in [Ca²⁺] _i and ROS in cardiac myocytes are induced by two parallel pathways initiated at the plasma membrane: One being the ouabain-altered transient interactions of a fraction of the Na⁺/K⁺-ATPase with neighboring proteins (Src, growth factor receptors, adaptor proteins, and Ras) leading to ROS generation, and the other, inhibition of the transport function of another fraction of the Na⁺/K⁺-ATPase leading to rise in [Ca²⁺] _i . Evidently, the gene regulatory effects of ouabain in cardiac myocytes require the downstream collaborations of ROS and [Ca²⁺] _i .

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 Ca²⁺-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 α-actin and atrial natriuretic factor, repression of the gene of the α₃-subunit of Na⁺/K⁺-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-κB. 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 Ca²⁺ 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.

Recent studies have ascribed many non-pumping functions to the Na/K-ATPase. Here, we present experimental evidence demonstrating that over half of the plasma membrane Na/K-ATPase in LLC-PK1 cells is performing cellular functions other than ion pumping. This “non-pumping” pool of Na/K-ATPase, like the pumping pump, binds ouabain. Depletion of either cholesterol or caveolin-1 moves some of the “non-pumping” Na/K-ATPase into the pumping pool. Graded knock-down of the α1 subunit of the Na/K-ATPase eventually results in loss of this “non-pumping” pool while preserving the pumping pool. Our prior studies indicate that a loss of the non-pumping pool is associated with a loss of receptor function as evidenced by the failure of ouabain administration to induce the activation of Src and/or ERK. Therefore, our new findings suggest that a substantial amount of surface-expressed Na/K-ATPase, at least in some types of cells, may function as non-canonical ouabain-binding receptors.

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 Ca²⁺ concentration ([Ca²⁺]_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 α₁- and α₂-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 α-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 α₁-isoform and caveolin-3; and 3) 50–60% increases in caveolar Src and α₂-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na⁺/K⁺-ATPase to ERK1/2 and [Ca²⁺]_i are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and α₂-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

We have observed recently that experimental renal failure in the rat is accompanied by increases in circulating concentrations of the cardiotonic steroid, marinobufagenin (MBG), and substantial cardiac fibrosis. We performed the following studies to examine whether MBG might directly stimulate cardiac fibroblast collagen production. In vivo studies were performed using the 5/6th nephrectomy model of experimental renal failure (PNx), MBG infusion (MBG), PNx after immunization against MBG, and concomitant PNx and adrenalectomy. Physiological measurements with a Millar catheter and immunohistochemistry were performed. In vitro studies were then pursued with cultured isolated cardiac fibroblasts. We observed that PNx and MBG increased MBG levels, blood pressure, heart size, impaired diastolic function, and caused cardiac fibrosis. PNx after immunization against MBG and concomitant PNx and adrenalectomy had similar blood pressure as PNx but less cardiac hypertrophy, diastolic dysfunction, and cardiac fibrosis. MBG induced increases in procollagen-1 expression by cultured cardiac fibroblasts at 1 nM concentration. These increases in procollagen expression were accompanied by increases in collagen translation and increases in procollagen-1 mRNA without any demonstrable increase in procollagen-1 protein stability. The stimulation of fibroblasts with MBG could be prevented by administration of inhibitors of tyrosine phosphorylation, Src activation, epidermal growth factor receptor transactivation, and N-acetyl cysteine. Based on these findings, we propose that MBG directly induces increases in collagen expression by fibroblasts, and we suggest that this may be important in the cardiac fibrosis seen with experimental renal failure.