title: NMDA receptor activation increases free radical production through nitric oxide and NOX2.
abstract: Reactive oxygen species (ROS) and nitric oxide (NO) participate in NMDA receptor signaling. However, the source(s) of the ROS and their role in the increase in cerebral blood flow (CBF) induced by NMDA receptor activation have not been firmly established. NADPH oxidase generates ROS in neurons, but there is no direct evidence that this enzyme is present in neurons containing NMDA receptors, or that is involved in NMDA receptor-dependent ROS production and CBF increase. We addressed these questions using a combination of in vivo and in vitro approaches. We found that the CBF and ROS increases elicited by topical application of NMDA to the mouse neocortex were both dependent on neuronal NO synthase (nNOS), cGMP, and the cGMP effector kinase protein kinase G (PKG). In mice lacking the NADPH oxidase subunit NOX2, the ROS increase was not observed, but the CBF increase was still present. Electron microscopy of the neocortex revealed NOX2 immunolabeling in postsynaptic somata and dendrites that also expressed the NMDA receptor NR1 subunit and nNOS. In neuronal cultures, the NMDA-induced increase in ROS was mediated by NADPH oxidase through NO, cGMP and PKG. We conclude that NADPH oxidase in postsynaptic neurons generates ROS during NMDA receptor activation. However, NMDA receptor-derived ROS do not contribute to the CBF increase. The findings establish a NOX2-containing NADPH oxidase as a major source of ROS produced by NMDA receptor activation, and identify NO as the critical link between NMDA receptor activity and NOX2-dependent ROS production.
authors.affiliation: Division of Neurobiology, Weill Cornell Medical College, New York, New York 10021, USA.
authors.forename: Helene
authors.initials: H
authors.lastname: Girouard
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Eduardo F
authors.initials: EF
authors.lastname: Gallo
authors.forename: Josef
authors.initials: J
authors.lastname: Anrather
authors.forename: Ping
authors.initials: P
authors.lastname: Zhou
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Pickel
authors.forename: Costantino
authors.initials: C
authors.lastname: Iadecola
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001921:Brain / Q000166:cytology; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006152:Cyclic GMP / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018683:Excitatory Amino Acid Agents / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016253:Microscopy, Immunoelectron / Q000379:methods; D016202:N-Methylaspartate / Q000494:pharmacology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000172:deficiency / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000172:deficiency; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013569:Synapses / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D004791:Enzyme Inhibitors; D018683:Excitatory Amino Acid Agents; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D052248:Nitric Oxide Synthase Type I; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP