title: Activation of constitutive nitric-oxide synthase activity is an early signaling event induced by ionizing radiation.
abstract: Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.
authors.affiliation: Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298-0058, USA.
authors.forename: J Kevin
authors.initials: JK
authors.lastname: Leach
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
authors.forename: Rupert K
authors.initials: RK
authors.lastname: Schmidt-Ullrich
authors.forename: Ross B
authors.initials: RB
authors.lastname: Mikkelsen
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000528:radiation effects*; D006162:Guanylate Cyclase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011839:Radiation, Ionizing; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects*; D014162:Transfection; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516727:Ptpn11 protein, rat; D006162:Guanylate Cyclase