title: Regulation of brain glutamate metabolism by nitric oxide and S-nitrosylation.
abstract: Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS(-/-)) or neuronal nitric oxide synthase (nNOS(-/-)). In particular, nNOS(-/-) animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. (15)N-glutamine-based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS(-/-) mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys(373) and Cys(561) in wild-type and eNOS(-/-) mice, but not in nNOS(-/-) mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism.
authors.affiliation: Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Karthik
authors.initials: K
authors.lastname: Raju
authors.affiliation: Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Paschalis-Thomas
authors.initials: PT
authors.lastname: Doulias
authors.affiliation: Department of Biomedical and Health Informatics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Perry
authors.initials: P
authors.lastname: Evans
authors.affiliation: Division of Neurology, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Elizabeth N
authors.initials: EN
authors.lastname: Krizman
authors.affiliation: Division of Neurology, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Jackson
authors.affiliation: Division of Genetic and Metabolic Disease, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Oksana
authors.initials: O
authors.lastname: Horyn
authors.affiliation: Division of Genetic and Metabolic Disease, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Yevgeny
authors.initials: Y
authors.lastname: Daikhin
authors.affiliation: Division of Genetic and Metabolic Disease, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Ilana
authors.initials: I
authors.lastname: Nissim
authors.affiliation: Division of Genetic and Metabolic Disease, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Yudkoff
authors.affiliation: Division of Genetic and Metabolic Disease, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Itzhak
authors.initials: I
authors.lastname: Nissim
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Sharp
authors.affiliation: Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA. Division of Neurology, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA. Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Robinson
authors.affiliation: Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA. Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA. Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA 19104, USA. ischirop@mail.med.upenn.edu.
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002853:Chromatography, Liquid; D003545:Cysteine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D051381:Rats; D026403:S-Nitrosothiols / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D020543:Proteome; D026403:S-Nitrosothiols; D005973:Glutamine; D009569:Nitric Oxide; D018698:Glutamic Acid; C021315:S-nitrosocysteine; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D003545:Cysteine