title: Ammonia triggers exocytotic release of L-glutamate from cultured rat astrocytes.
abstract: Ammonia toxicity to the brain involves NMDA receptor overactivation and glutamate excitotoxicity. The mechanisms underlying glutamate release from astrocytes in response to ammonia were addressed in this study. In cultured rat astrocytes, glutamate immunoreactivity (IR) was punctate and partly colocalized with transfected VAMP2-YFP. NH(4)Cl (5 mmol/L) and hypoosmotic exposure (205 mosmol/L) induced a rapid colchicine-sensitive loss of cellular glutamate and glutamate appearance in the extracellular space. The NH(4)Cl-induced glutamate loss from astrocytes was strongly blunted after transfection of the cells with VAMP2 siRNA. Ammonia-induced exocytosis of VAMP2-YFP expressing vesicles was shown by total internal reflection fluorescence microscopy (TIRF-M). Glutamate exocytosis in response to ammonia was sensitive to chelation of Ca(2+), cyclooxygenase inhibition by indomethacin and colchicine. Ammonia triggered the rapid formation of prostanoids, which were identified as upstream events in ammonia-induced glutamate exocytosis. Also, addition of prostaglandin E(2) or of tumor necrosis factor (TNF)-alpha triggered glutamate exocytosis. Inhibition of ammonia-induced glutamate exocytosis after transfection of VAMP2 siRNA inhibited ammonia-induced RNA oxidation. It is concluded that ammonia triggers a prostanoid- and Ca(2+)-dependent glutamate exocytosis, which is essential for induction of ammonia-induced RNA oxidation.
authors.affiliation: Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich Heine University, Düsseldorf, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Görg
authors.forename: Anke
authors.initials: A
authors.lastname: Morwinsky
authors.forename: Verena
authors.initials: V
authors.lastname: Keitel
authors.forename: Natalia
authors.initials: N
authors.lastname: Qvartskhava
authors.forename: Karsten
authors.initials: K
authors.lastname: Schrör
authors.forename: Dieter
authors.initials: D
authors.lastname: Häussinger
mesh_terms: D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D001921:Brain / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D005470:Fluorometry / Q000379:methods; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D007213:Indomethacin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D011453:Prostaglandins / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection / Q000379:methods; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D016861:Cyclooxygenase Inhibitors; C086686:FM2 10; D005904:Glial Fibrillary Acidic Protein; D008164:Luminescent Proteins; D011453:Prostaglandins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C054562:yellow fluorescent protein, Bacteria; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D018698:Glutamic Acid; D004533:Egtazic Acid; D012313:RNA; D000641:Ammonia; D002118:Calcium; D007213:Indomethacin