Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPase to hypertrophy and its marker genes in cardiac myocytes

Departments of Pharmacology and Medicine, Medical College of Ohio, Toledo, Ohio 43614, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 08/1999; 274(27):19323-8. DOI: 10.1074/jbc.274.27.19323
Source: PubMed

ABSTRACT 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 alpha-actin and atrial natriuretic factor, repression of the gene of the alpha3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-kappaB. 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.

Download full-text


Available from: Joseph I Shapiro, Jul 06, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cardiotonic steroids have been used for the past 200 years in the treatment of congestive heart failure. As specific inhibitors of membrane-bound Na+/K+ ATPase, they enhance cardiac contractility through increasing myocardial cell calcium in response to the resulting increase in intracellular Na. The half-minimal concentrations of cardiotonic steroids required to inhibit Na+/K+ ATPase range in nM-uM concentrations. In contrast, the circulating levels of cardiotonic steroids under physiologic conditions are in the low picomolar concentration range in healthy individuals, increasing to high picomolar levels under pathophysiologic conditions including chronic kidney disease and heart failure. Little is known about the physiologic function of low picomolar concentrations of cardiotonic steroids. Recent studies suggest that physiologic concentrations of cardiotonic steroids acutely stimulate the activity of Na+/K+ ATPase and activate an intracellular signaling pathway that regulates a variety of intracellular functions including cell growth and hypertrophy. The effect of circulating cardiotonic steroids on renal salt handling and total body sodium homeostasis is unknown. This review will focus on the role of low picomolar concentrations of cardiotonic steroids on renal Na+/K+ ATPase activity, cell signaling, and blood pressure regulation.
    Journal of Endocrinology 04/2014; DOI:10.1530/JOE-13-0613 · 3.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have shown that the Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H2O2-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H2O2 generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPase and in ouabain-sensitive Rb(+) uptake. These effects of H2O2 appear to be similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H2O2 on Src and ERK1/2 were inhibited in the α1 Na/K-ATPase knockdown PY-17 cells. While expression of wild type α1 or A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1 but not A420P mutant, was able to restore the H2O2-induced activation of protein kinases. Consistently, disrupting the formation of Na/K-ATPase/Src complex by pNaKtide attenuated effects of H2O2 on the kinases. Moreover, a direct effect of H2O2 on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H2O2 reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggests that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H2O2 to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.
    Free Radical Biology and Medicine 04/2014; 71. DOI:10.1016/j.freeradbiomed.2014.03.036 · 5.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ammonia toxicity is clinically important and biologically poorly understood. We reported previously that 3 mM ammonia chloride (ammonia), a relevant concentration for hepatic encephalopathy studies, increases production of endogenous ouabain and activity of Na,K-ATPase in astrocytes. In addition, ammonia-induced upregulation of gene expression of α2 isoform of Na,K-ATPase in astrocytes could be inhibited by AG1478, an inhibitor of the EGF receptor (EGFR), and by PP1, an inhibitor of Src, but not by GM6001, an inhibitor of metalloproteinase and shedding of growth factor, suggesting the involvement of endogenous ouabain-induced EGF receptor transactivation. In the present study, we investigated ammonia effects on phosphorylation of EGF receptor and its intracellular signal pathway towards MAPK/ERK1/2 and PI3K/AKT; interaction between EGF receptor, α1, and α2 isoforms of Na,K-ATPase, Src, ERK1/2, AKT and caveolin-1; and relevance of these signal pathways for ammonia-induced cell swelling, leading to brain edema, an often fatal complication of ammonia toxicity. We found that i) ammonia increases EGF receptor phosphorylation at EGFR(845) and EGFR(1068); ii) ammonia-induced ERK1/2 and AKT phosphorylation depends on the activity of EGF receptor and Src, but not on metalloproteinase; iii) AKT phosphorylation occurs upstream of ERK1/2 phosphorylation; iv) ammonia stimulates association between the α1 Na,K-ATPase isoform, Src, EGF receptor, ERK1/2, AKT and caveolin-1; v) ammonia-induced ROS production might occur later than EGFR transactivation; vii) both ammonia induced ERK phosphorylation and ROS production can be abolished by canrenone, an inhibitor of ouabain, and vi) ammonia-induced cell swelling depends on signaling via the Na,K-ATPase/ouabain/Src/EGF receptor/PI3K-AKT/ERK1/2, but in response to 3 mM ammonia it does not appear until after 12 hr. Based on literature data it is suggested that the delayed appearance of the ammonia-induced swelling at this concentration reflects required ouabain-induced oxidative damage of the ion and water cotransporter NKCC1. This information may provide new therapeutic targets for treatment of hyperammonic brain disorders.
    Neurochemistry International 09/2013; DOI:10.1016/j.neuint.2013.09.005 · 2.65 Impact Factor