title: Threshold levels of extracellular l-arginine that trigger NOS-mediated ROS/RNS production in cardiac ventricular myocytes.
abstract: l-Arginine (L-Arg) is the substrate for nitric oxide synthase (NOS) to produce nitric oxide (NO), a signaling molecule that is key in cardiovascular physiology and pathology. In cardiac myocytes, L-Arg is incorporated from the circulation through the functioning of system-y+cationic amino acid transporters. Depletion of L-Arg leads to NOS uncoupling, with O2rather than L-Arg as the terminal electron acceptor, resulting in superoxide formation. The reactive oxygen species (ROS) superoxide (O2˙-), combined with NO, may lead to the production of the reactive nitrogen species (RNS) peroxynitrite (ONOO-), which is recognized as a major contributor to myocardial depression. In this study we aimed to determine the levels of external L-Arg that trigger ROS/RNS production in cardiac myocytes. To this goal, we used a two-step experimental design in which acutely isolated cardiomyocytes were loaded with the dye coelenterazine that greatly increases its fluorescence quantum yield in the presence of ONOO-and O2˙-Cells were then exposed to different concentrations of extracellular L-Arg and changes in fluorescence were followed spectrofluorometrically. It was found that below a threshold value of ~100 µM, decreasing concentrations of L-Arg progressively increased ONOO-/ O2˙--induced fluorescence, an effect that was not mimicked by d-arginine or l-lysine and was fully blocked by the NOS inhibitor l-NAME. These results can be explained by NOS aberrant enzymatic activity and provide an estimate for the levels of circulating L-Arg below which ROS/RNS-mediated harmful effects arise in cardiac muscle.
authors.affiliation: Department of Pharmacology, Physiology and Neuroscience, Rutgers Biomedical and Health Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey.
authors.forename: Jayalakshmi
authors.initials: J
authors.lastname: Ramachandran
authors.affiliation: Department of Pharmacology, Physiology and Neuroscience, Rutgers Biomedical and Health Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey dpeluffo@unorte.edu.uy.
authors.forename: R Daniel
authors.initials: RD
authors.lastname: Peluffo
mesh_terms: D000818:Animals; D001120:Arginine / Q000008:administration & dosage*; D002478:Cells, Cultured; D004056:Differential Threshold / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D045604:Extracellular Fluid / Q000187:drug effects / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D001120:Arginine; D019001:Nitric Oxide Synthase
keywords: cationic amino acid transporters; nitric oxide; peroxynitrite; superoxide