title: Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle.
abstract: Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% ("physiologic") or 20% ("supraphysiologic") at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Dick
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8.
authors.forename: Julijana
authors.initials: J
authors.lastname: Ivanovska
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8.
authors.forename: Crystal
authors.initials: C
authors.lastname: Kantores
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8.
authors.forename: Rosetta
authors.initials: R
authors.lastname: Belcastro
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8; Department of Physiology, Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: A
authors.initials: A
authors.lastname: Keith Tanswell
authors.affiliation: Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 1X8; Department of Physiology, Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Division of Neonatology, Department of Paediatrics, and Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8; Heart and Stroke Richard Lewar Centre of Excellence, University of Toronto, Toronto, ON, Canada M5S 1A8. Electronic address: robert.jankov@sickkids.ca.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Jankov
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000502:physiology; D052248:Nitric Oxide Synthase Type I / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000096:biosynthesis*; D011651:Pulmonary Artery / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013314:Stress, Mechanical
chemical_list: D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat
keywords: Free radicals; NADPH oxidase; NOX4; Peroxynitrite; Smooth muscle; Stretch