Ammonia triggers exocytotic release of L-glutamate from cultured rat astrocytes.

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.