title: Ouabain increases iNOS-dependent nitric oxide generation which contributes to the hypertrophic effect of the glycoside: possible role of peroxynitrite formation.
abstract: In addition to inotropic effects, cardiac glycosides exert deleterious effects on the heart which limit their use for cardiac therapeutics. In this study, we determined the possible contribution of ouabain-induced iNOS stimulation to the resultant hypertrophic as well as cytotoxic effects of the glycoside on cultured adult rat ventricular myocytes. Myocytes were treated with ouabain (50 μM) for up to 24 h. Ouabain significantly increased gene and protein levels of inducible nitric oxide synthase (iNOS) which was associated with significantly increased release of NO from myocytes as well as increased total release of reactive oxygen species (ROS), superoxide anion (O(2) (-)), and increased peroxynitrite formation as assessed by protein tyrosine nitration. Administration of ouabain was also associated with increased levels of myocyte toxicity as determined by myocyte morphology, trypan blue staining and lactate dehydrogenase (LDH) efflux. The nonspecific NOS inhibitor Nω-nitro-L: -arginine methyl ester and the more selective iNOS inhibitor 1400W both abrogated the increase in LDH release but had no significant effect on either morphology or trypan blue staining. Ouabain also significantly increased both myocyte surface area and expression of atrial natriuretic peptide indicating a hypertrophic response with both parameters being completely prevented by NOS inhibition. The effects of iNOS inhibitors were associated with diminished ouabain tyrosine nitration as well as abrogation of ouabain-induced p38 and ERK phosphorylation. Our study shows that ouabain is a potent inducer of NO formation, iNOS upregulation, and increased production of ROS. Inhibition of ouabain-dependent peroxynitrite formation may contribute to the antihypertrophic effect of iNOS inhibition possibly by preventing downstream MAPK activation.
authors.affiliation: Department of Physiology & Pharmacology, Schulich School of Medicine and Dentistry,  University of Western Ontario, London, ON, N6A 5C1, Canada.
authors.forename: Xiaohong Tracey
authors.initials: XT
authors.lastname: Gan
authors.forename: J Craig
authors.initials: JC
authors.lastname: Hunter
authors.forename: Cathy
authors.initials: C
authors.lastname: Huang
authors.forename: Jenny
authors.initials: J
authors.lastname: Xue
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Rajapurohitam
authors.forename: Sabzali
authors.initials: S
authors.lastname: Javadov
authors.forename: Morris
authors.initials: M
authors.lastname: Karmazyn
mesh_terms: D000818:Animals; D002316:Cardiotonic Agents / Q000633:toxicity*; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D017379:Hypertrophy, Left Ventricular / Q000139:chemically induced* / Q000201:enzymology / Q000473:pathology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010042:Ouabain / Q000633:toxicity*; D030421:Peroxynitrous Acid / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D010042:Ouabain; D007770:L-Lactate Dehydrogenase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases